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Development 120, 3379-3394 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 3379 Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras Laure Bally-Cuif and Marion Wassef INSERM U106, Hôpital de la Salpêtrière, 47 Bd de l’hôpital, 75651-Paris Cedex 13, France and CNRS URA 1414, Equipe ATIPE, Ecole Normale Supérieure, 46 rue d’Ulm, 75230-Paris Cedex 05, France (present address) SUMMARY When grafted ectopically into the diencephalon of a chick host embryo, a portion of met-mesencephalon straddling the met-mesencephalic constriction has the capacity to induce En-2 expression in the surrounding host tissue. Subsequently, tectal and cerebellar structures, composed of both host and grafted cells, are reconstructed in this ectopic location at the expense of the host diencephalon. Previous experiments indicated that the induction of En-2 was correlated with Wnt-1 expression within the graft. The aim of the present study was: (i) to determine whether Wnt-1 expression was spatially regulated within the graft, (ii) to investigate whether host Wnt-1-expressing cells were also involved in the ectopic met-mesencephalic development and, if so, (iii) to localize these Wnt-1-positive domains in relation to the patterning of the ectopically developing met-mesencephalic territory. We studied the expression profile of Wnt1, in relation with that of other positional markers, in quail/chick chimeras where various portions of met-mesencephalon had been grafted into the diencephalon. We found that Wnt-1 expression was reorganized within the graft, and that it was also induced in the host in contact with the graft. Moreover, these ectopic expressions of Wnt-1, in both the grafted and the surrounding host tissues, were organized in concert to form a continuous positive line at the host/graft junction, the location of which depended on the precise origin of the graft. Finally, we found that this line was frequently located at the limit between territories expressing different positional markers. We propose that Wnt-1 expression is turned on at the junction between domains of different phenotypes, and may be used as a border to stabilize these adjacent differently committed territories. INTRODUCTION A good candidate for the specification step is the product of the Wnt-1 gene (Fung et al., 1985), the homolog of the Drosophila wingless protein (Rijsewick et al., 1987), a secreted protein involved in cell-cell signaling (Bradley and Brown, 1990; Papkoff and Schryver, 1990; Jue et al., 1992). Wnt-1 is known to be expressed in the developing met-mesencephalon of all vertebrate species studied (mouse (Wilkinson et al., 1987), Xenopus (Noordermeer et al., 1989), and zebrafish (Molven et al., 1991)). In the mouse embryo, expression in this region is very dynamic (Wilkinson et al., 1987; Bally-Cuif et al., 1992; Paar et al., 1993): it starts at the one-somite stage as a broad domain encompassing what is probably most of the met-mesencephalic region, and is rapidly restricted after neural tube closure to a narrow ring of cells, located just rostrally to the morphological constriction that separates the mesencephalic and metencephalic vesicles. Disruption of the Wnt-1 locus in the mouse, either by homologous recombination or in the spontaneous mutation swaying (Thomas et al., 1991) leads, since early embryonic stages, to severe brain defects, characterized by the lack of a broad domain encompassing most of the presumptive mesencephalon and cerebellum (McMahon and Bradley, 1990; Thomas and Capecchi, 1990; McMahon et al., 1992). For that reason, Wnt-1 is believed to play a crucial role in the early development of the met-mesencephalic region. The so-called met-mesencephalic domain of the embryonic neural tube, which comprises the mesencephalic and the first rhombencephalic vesicles, is the region from which derive the adult mesencephalon (colliculi in the mouse, optic tectum in the chick) and cerebellum. Although these adult structures are clearly distinct both in morphology and in function, the embryonic met-mesencephalon seems to evolve as a unified region. In particular, this entire domain, but no other region of the neural tube, specifically expresses the homeobox-containing genes of the engrailed class, En-1 and En-2, since early stages of neurulation (Davis and Joyner, 1988; Davis et al., 1988; Gardner et al., 1988, Gardner and Barald, 1992). Moreover, when grafted ectopically into the diencephalon of a chick host embryo, portions of this domain are able to induce in the contacted host tissue the ectopic expression of En-2 (Gardner and Barald, 1991; Martinez et al., 1991) and the development of the missing met-mesencephalic parts (Martinez et al., 1991). The mesencephalic and metencephalic vesicles therefore seem to evolve in concert, at least at early stages. How this domain is initially specified as a whole, and later subdivided into its mesencephalic and cerebellar components, is not yet known. Key words: Wnt-1, met-mesencephalon, quail/chick chimeras, boundary formation 3380 L. Bally-Cuif and M. Wassef These studies on mutant mice (McMahon et al., 1992) have also identified En-1 as a gene whose early maintenance of expression in mesencephalon was dependent on Wnt-1; but later-occurring regulatory interactions in met-mesencephalon could not be studied since the whole En-expressing domain rapidly disappears. In particular, what role Wnt-1 plays after its expression domain has been restricted to a narrow ring is not known. We and others have been studying the organization and early development of the met-mesencephalic region in the chick embryo in normal and experimentally manipulated animals, exploiting the ease with which tissue transplantation is feasible in this species (Martinez and Alvarado-Mallart, 1989; 1990; Hallonet et al., 1990; Gardner and Barald, 1991; Martinez et al., 1991). Using mouse/chick chimeras, we were able to show that the Wnt-1-positive portion of mouse met-mesencephalon always maintains its Wnt-1 expression when grafted ectopically, and is the region with the highest capacity to induce ectopic expression of En-2 in the diencephalon of the chick host embryo (Bally-Cuif et al., 1992). Again, these results suggested that an early Wnt-1 expression might be implicated in the specification of the met-mesencephalic domain. The expression pattern of Wnt-1 in these grafts was not precisely analyzed but seemed to be reorganized at the periphery of the grafted tissue. Whether the normal pattern of Wnt-1 expression was also modified in the chick host, and in particular whether host Wnt-1-expressing cells were also participating in the development of the ectopic met-mesencephalon, could however not be determined due to the lack of a probe recognizing chick Wnt-1 transcripts. Similarly, whether Wnt-1 expression in and around the ectopic met-mesencephalon was spatially organized, relative to other positional markers, was not studied. The aim of the present study was to explore these possibilities. We have cloned a portion of the chick Wnt-1 cDNA and used it, together with the corresponding quail Wnt-1 cDNA fragment (a kind gift of Marc Hallonet), to study Wnt-1 regulation in both the host and the grafted tissue during an ectopic met-mesencephalic development. Our results indicate that the host and the graft frequently cooperate to reconstruct a continuous line of Wnt-1-expressing cells at the host/graft junction, which, at least in the case of metencephalic grafts, separates two territories of different phenotypes. We hypothesize that Wnt-1-expressing cells serve as a boundary to stabilize these grafted metencephalic portions within the surrounding tissue. MATERIALS AND METHODS Embryos We used White Leghorn chick embryos (Haas, Strasbourg, France), and Japanese quail embryos (La Caille de Chanteloup, Corps-Nuds, France), staged according to Hamburger and Hamilton (1951). Cloning of 403 bp of the chick Wnt-1 cDNA We used RT-PCR and degenerate oligonucleotides (designed by comparing the Wnt-1 cDNA sequences of mouse (Fung et al., 1985), Xenopus (Noordermeer et al., 1989) and zebrafish (Molven et al., 1991)) to amplify a 403 bp fragment of the coding region. The position of the oligonucleotides relative to the mouse sequence is presented in Fig. 1. ClaI and XbaI restriction sites added at the 5′ end of oligonucleotides a and b, respectively, are underlined. The sequences are as follows: oligo a: 5′ CCATCGATTCTGGTGGGG(C,T,G)AT(C,T,A)GT 3′ oligo b: 5′ GCTCTAGATACCCAGTGCCAGTCGGG 3′ oligo c: 5′ CAT(C,T)CC(G,A)TG(G,A)CA(C,T)TTGCA 3′ Total RNA was isolated from HH10-12 chick embryos by guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987). 10 µg RNA were then reverse transcribed with 50 u of MMLV H− reverse transcriptase (Superscript, BRL), 200 pmoles random primers (Boehringer Mannheim) and 1 mM each dNTP in 20 µl MMLV buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 20 mM DTT) for 1 hour at 42°C. After heating at 95°C and ethanol precipitation, cDNA prepared from 10 µg of RNA was used in a PCR reaction (Perkin Elmer Cetus) with 100 pmoles primer a, 100 pmoles primer c and 2.5 u Taq DNA polymerase (Promega) in 50 mM KCl, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM each dNTP, according to the following schedule: 94°C for 1 minute, 40°C for 1 minute and 72°C for 1 minute per cycle for the two first cycles, then 94°C for 1 min., 52°C for 1 minute and 72°C for 1 minute per cycle for the 28 following cycles. One thousandth of the total PCR reaction mix was then amplified again using the same stringency conditions but with 100 pmoles primer b instead of primer c. The final reaction product was run on a 2% agarose gel, the band at 403 bp was excised and purified by phenol-chloroform extraction and ethanol precipitation. The fragment was then digested by XbaI and ClaI according to the manufacturer’s instructions and subcloned into pBluescript KS(+) (Stratagene). The fragment was sequenced on both strands using T7 DNA polymerase (Sequenase version 2.0, U.S. Biochemical Corp.). Probes The Ch.Wnt-1 subclone (Fig. 1) was linearized with ClaI and transcribed using T7 RNA polymerase, or linearized with XbaI and transcribed with T3 RNA polymerase, to generate the antisense and sense probes, respectively. The Pax QNR (Pax 6) subclone (Martin et al., 1992) was linearized with EcoRI and transcribed using T7 RNA polymerase; the Cotx2 subclone (Bally-Cuif et al., in press) was linearized with EcoRI and transcribed with T3 RNA polymerase; and the Q.Wnt1 subclone (kind gift of Marc Hallonet) was linearized with NotI and transcribed with SP6 RNA polymerase, to generate the other antisense probes. No signals were obtained with the corresponding sense probes. Grafting experiments The experimental designs are presented in Fig. 2. After a small opening was made in the egg, HH10-11 chick host embryos were visualized by a sub-blastodermal injection of India ink, the vitelline membrane was cut, and a small portion of the right-hand side of mesencephalon (A) or diencephalon (B) was removed using a bevelled needle. Quail donor embryos of the same stage were removed from the egg and pinched in a paraffin-containing Petri dish in Tyrodes saline solution. The desired portion of met-mesencephalon, either from the right (non inverted grafts) or from the left (inverted grafts) side of the embryo, was then cut out and transported to the chick host with a glass pipette, to replace the ablated portion of mesencephalon or diencephalon. The dorsolateral orientation of the graft was always respected, so that pieces taken from the left side of the embryo were grafted with a rostrocaudal inversion. The egg was then closed with parafilm and returned to the incubator for 48 to 72 hours after which time the embryos were fixed and processed for whole-mount in situ hybridization. Single colour whole-mount in situ hybridization Embryos were fixed by immersion overnight at 4°C in 4% paraformaldehyde and dehydrated in methanol. For embryos older than HH10, the neural tube was manually dissected after 1-2 hours of Wnt-1 in met/mesencephalic grafts 3381 Fig. 1. (A) Position of the Ch.Wnt-1 amplified fragment (b.) relative to the mouse Wnt-1 coding sequence (a.) (Fung et al., 1985). The primers a, b and c used in the PCR reaction are indicated (arrows). Size of cDNA fragments are in bp. (B) Alignment of mouse Wnt-1 (Fung et al., 1985) and Ch.Wnt-1 nucleotide sequences (above), and the corresponding amino acid sequences of mouse Wnt-1, Wnt-2, Wnt-3, Wnt-4 (Gavin et al., 1990) with Ch. Wnt-1 (below). Dashes and asterisks indicate identical residues or gaps, respectively. The Q.Wnt-1 corresponding fragment shows 84% sequence identity with Ch.Wnt-1 at the nucleotide level, and 96% at the protein level (Hallonet, 1993). fixation. Whole-mount in situ hybridization was done exactly as described (Bally-Cuif et al., 1993) except that the anti-digoxigeninalkaline phosphatase (AP) antibody was diluted 1/2000. When two different mRNAs were detected, the two corresponding probes were labeled with digoxigenin-UTP, added together at the same concentration (1-2 µg/ml) to the hybridization buffer and revealed using NBT/BCIP as a substrate for alkaline phosphatase. After the colour reaction, embryos were rinsed extensively in PBS-0.1% Tween-20 (PBT) and stored, photographed and flat-mounted in 80% glycerol in PBT. Staining was never observed with any sense control probe. In addition, no signal was ever observed, under our conditions, in chick embryos using the Q.Wnt-1 probe and vice-versa. In fact, sequence homology at the nucleotide level was not any higher between chick and quail than between chick and mouse Wnt-1, which do not crossreact (Bally-Cuif et al., 1992). Double colour whole-mount in situ hybridization Riboprobes were synthesized with incorporation of digoxigenin-UTP or fluorescein-UTP (both from Boehringer Mannheim) in the same conditions. Embryos were fixed, pretreated and prehybridized as previously described (Bally-Cuif et al., 1993). The two probes were added to the hybridization buffer at the same concentration (1-2 µg/ml) and hybridization was performed overnight at 70°C. Posthybridization washes were as described (Bally-Cuif et al., 1993), and embryos were preblocked in 10% normal goat serum (Gibco) in TBST (1.5 mM NaCl, 0.03 mM KCl, 0.025 M Tris-HCl pH 7.5, 0.1% Tween 20) for 2 hours at room temperature. They were then incubated in either anti-digoxigenin-AP antibody (1/2000, Boehringer Mannheim) or in anti-fluorescein-AP antibody (1/500, Boehringer Mannheim), depending on which probe was to be revealed first, both in 1% NGS in TBST-2 mM levamisole overnight at 4°C. Rinses were in TBST2 mM levamisole, and AP activity was revealed using NBT/BCIP as described (Bally-Cuif et al., 1993). After the colour had developed, the embryos were extensively rinsed in PBT and incubated overnight at 4°C in anti-fluorescein-AP (1/500) or anti-digoxigenin-AP (1/2000), depending on the second probe to be revealed, in 1% NGS in TBST-2 mM levamisole. They were then rinsed for 5 hours in many changes of TBST-2 mM levamisole. AP activity was then revealed using Fast Red (Bioprobe Systems), as follows: embryos were preincubated in Naphtol phosphate buffer (Bioprobe Systems)-0.1% Tween-20 for 30 minutes, then in the same buffer containing 3 mg/ml Fast Red powder, in the dark and in glass containers, until red colour developed. Residual alkaline phosphatase activity from the first reaction was generally negligible, and did not interfere with the second colour development (see in particular Fig. 5, where the two colors are clearly distinct). Whole-mount immunocytochemistry The chick or quail En-2 protein was revealed using the 4D9 monoclonal antibody (Patel et al., 1989). Immunostaining was always performed after in situ hybridization. After AP activity had been revealed, the embryos were extensively rinsed in PBT, and incubated in 4D9 antibody diluted to 1/2 in PBS-2 g/l gelatin-0.25% Triton X- 3382 L. Bally-Cuif and M. Wassef Fig. 2. Schematic representation of the grafting experiments. (A) Homotopic mesencephalic grafts: a medial portion of the right side of the mesencephalic vesicle (black) was ablated from a HH11 chick embryo, and grafted into a HH11 quail host. Grafted embryos were analyzed at HH14 for Ch.Wnt-1 expression. (B) Heterotopic grafts: one side of the mesencephalic (A), metencephalic (B) or met-mesencephalic (C1,C2,C3) domains (stripes) was ablated from a HH11 quail embryo and grafted into the diencephalon of a HH11 chick host. The dorsal midline region present in the graft was always put in contact with that of the host neural tube, so that fragments taken from the left side of the quail embryo were grafted in a rostrocaudally inverted orientation (curved arrow). Embryos were analyzed at HH18-20. 100 (PGT) overnight at 4°C. Subsequent rinses were in PT (PBS0.025% Triton X-100), and the antibody was revealed using the peroxidase-anti-peroxidase method of Sternberger et al. (1970). Cryostat sectioning of embryos and propidium iodide counterstaining After whole-mount in situ hybridization, embryos were cryoprotected in 15% sucrose in phosphate buffer. They were then embedded in 7.5% gelatin in 15% sucrose in phosphate buffer (Canning and Stern, 1988), frozen in isopentane in liquid nitrogen at −50°C, and cryostat sectioned at 15 µm. Sections were degelatinized for 30 minutes in PBS at 37°C, and counterstained with propidium iodide (see Fig. 5F,G) by incubation for 30 minutes at room temperature in a 1 µg/ml solution. Sections were then mounted in Mowiol (Calbiochem), and quail nuclei were identified by the presence of a fluorescent red spot corresponding to their nucleolus-associated heterochromatin. RESULTS Cloning of a 403 bp fragment of the chick Wnt-1 cDNA We used degenerate oligonucleotides and two rounds of RTPCR to amplify a 403 bp cDNA fragment of the chick Wnt-1 gene (Ch.Wnt-1), corresponding to nt 285-689 of the mouse Wnt-1 cDNA sequence (Fung et al., 1985; see Fig. 1A). After one round of amplification using oligonucleotides a and c, a fragment of correct size was obtained, but additional bands, corresponding to non-specific hybridization of the primers, were also visible. Reamplification at this stage with a nested 3′ primer (b) gave a single 403 bp fragment which corresponded in size to the cognate mouse sequence (see Fig. 1A). This fragment was purified, subcloned into pBluescript KS(+) and sequenced on both strands. The sequences of two subclones obtained from two independent RT-PCR reactions were determined and proved to be identical. The sequence of the 403 bp fragment is given in Fig. 1B, aligned with the corresponding region of the mouse Wnt-1 cDNA (Fung et al., 1985). The sequences are 84% identical at the nucleotide level, and 92% at the protein level. As expected, this homology is slightly higher than that observed between the mouse and Xenopus (Noordermeer et al., 1989) or between the mouse and zebrafish (Molven et al., 1991) sequences. Most of the amino acid exchanges occurred in the same region of the protein in these four species, indicating that they may not be of functional importance. The sequence comparisons (Fig. 1B), together with the expression pattern reported below, strongly suggest that we have indeed isolated part of the chick homolog of Wnt-1. We have therefore used this cDNA fragment as a probe to study the spatial regulation of Wnt-1 expression during development of the chick embryonic neural tube, both in the normal embryo and after experimental manipulations. Wnt-1 in met/mesencephalic grafts 3383 Expression pattern of the Wnt-1 gene during normal development of the chick embryonic neural tube In a first set of experiments, we determined the spatiotemporal evolution of Wnt-1 expression in the chick embryonic neural tube, between stages HH1 and HH35 (embryonic day 8 [E8]), by whole-mount in situ hybridization (ISH). Control experiments at various stages on entire embryos (that is, without dissecting away non-neural tissues) showed that Wnt1 expression was, as reported for the other species studied, confined to the developing neural tube. No Wnt-1 expression was detected prior to the condensation of the first somite (HH7, 24 hour incubation), that is several hours after the beginning of neural plate formation. Wnt-1 transcripts first appear at HH7, on the dorsal region of the anterior neural folds, overlapping the sites of neural tube closure (Fig. 3A). At HH9 (7 somites), this expression domain has extended posteriorly and anteriorly, and additional positive cells have also started to appear on the lateral walls of the neural tube (hardly visible on Fig. 3B), in a region that, based on the pattern observed at later stages (see below at E2), we identify as the presumptive mesencephalic vesicle. Later (HH11-12, E2, 10-15 somites), expression is detected on the dorsal midline throughout the length of the neural tube up to the presumptive diencephalon (Fig. 3C,D), but the intensity is not uniform, and weakly positive regions in the spinal cord and metencephalic primordia separate two sharply delimited regions of intense staining: the mesencephalic vesicle and the rhombic lips of rhombomere 4. Most of the mesencephalic vesicle is labelled at this stage (Fig. 3C). The staining is more intense in the alar than in the basal plate, except near the met-mesencephalic constriction where it forms a large ring encircling the neural tube (Fig. 3D). Digoxigenin-labelled probes offer single cell resolution, and it is obvious at higher magnification (Fig. 4G) that the intensity of expression in this domain is uneven: small clusters of strongly Wnt-1-positive cells are juxtaposed to clusters of faintly positive or negative cells. This was already suggested in the E8.5 mouse (McMahon et al., 1992). A few patches of strongly positive cells are also detected in the dorsal region of the metencephalic vesicle (see Fig. 4G). At stage HH12-13 (1619 somites), Wnt-1 expression transiently extends anteriorly into the diencephalic vesicle, which now starts to be visible (Fig. 3E). The met-mesencephalic ring remains however the most strongly positive region. At E3 (HH14-15, when the neural tube is entirely closed in its caudal part, and the characteristic diencephalic and telencephalic vesicles have developed) the Wnt-1-positive territory is confined to a ring of cells, encircling the neural tube except for its ventral midline, immediately rostral to the constriction separating the met- and mesencephalic vesicles (Fig. 3F,G). Wnt-1 expression therefore disappears very rapidly from the anterior mesencephalon between HH12-13 and HH14. At E3, Wnt-1 expression is also clearly detected on the dorsal midline of the neural tube, both caudally throughout the length of the presumptive spinal cord, up to the edge of the cerebellar plate (the dorsal midline of which is now negative), and rostrally in the mesencephalic and part of the diencephalic vesicles, up to the epiphysis. Expression in the latter domain seems on wholemount stainings to consist of two parallel rows of stained cells (Fig. 3G,H, arrows). A sharp transition from strong to weak signal intensity is visible between the mesencephalon and diencephalic vesicles, respectively (Fig. 3H). The same two domains of expression are found at later stages. Expression on the edge of the cerebellar anlage progressively increases. Until E7 (HH31-32, see Fig. 3K for a E6 (HH28) embryo), the Wnt-1-positive ring encircling the neural tube (except for the ventral midline) remains located slightly rostrally to the met-mesencephalic constriction. Starting at E8, expression in the met-mesencephalic ring is progressively turned off, beginning with its lateral parts (not shown). Expression on the dorsal midline at this stage remains intense. The spatiotemporal pattern of Ch.Wnt-1 expression on the dorsal midline of the neural tube and in the met-mesencephalic region appears similar to that observed for the mouse Wnt-1 gene at equivalent stages (Wilkinson et al., 1987; Bally-Cuif et al., 1992; McMahon et al., 1992; Parr et al., 1993). However, a third domain of expression was detected on the ventral midline of the mesencephalon and part of the diencephalon in the mouse embryo between E9.5 and at least E12.5, which we did not see in the chick (compare Fig. 2F and I), even when using radioactive probes (not shown). The study of Wnt-1 expression in quail embryos using a quail Wnt-1 probe (Q.Wnt1) reveals the same pattern as described in the chick at equivalent stages (not shown). As in other species, Wnt-1 expression in chick and quail embryos thus appears to be closely associated with the early development of the met-mesencephalic region of the neural tube. The avian embryo lends itself to experimental manipulations and we have subsequently analyzed the regulation of Wnt-1 expression during the development of portions of metmesencephalon that were homotopically or heterotopically grafted in a chick host neural tube. The spatial restriction of Wnt-1 expression between HH12-13 and HH14 results from down-regulation of the gene rather than from caudal migration of positive cells Between HH12-13 and HH14, the met-mesencephalic domain is the site of important cell migrations and morphogenetic movements. To investigate whether the disappearance of Wnt1 expression in mesencephalon after stage HH13 was due to the migration of positive cells towards the met-mesencephalic ring, we performed the grafting experiments presented in Fig. 2A. The rostral mesencephalon of a HH11 chick embryo was grafted homotopically into a quail embryo of the same stage and analyzed for Ch.Wnt-1 expression at HH14 (quail Wnt-1 transcripts are not detected with the chick Wnt-1 probe). In all cases analyzed (n=13), Ch.Wnt-1-positive cells were only detected along the dorsal midline and not at the level of the met-mesencephalic ring (Fig. 3J), indicating that the grafted Wnt-1-positive cells did not migrate caudally to participate in the Wnt-1 ring. Rather, the spatial restriction of the Wnt-1positive domain must result from the down-regulation of the gene in most of the mesencephalic vesicle. The subsequent series of experiments were aimed at analyzing the regulation of Wnt-1 expression in portions of met-mesencephalon which were ectopically grafted into more anterior regions of the neural tube, in relation with the patterning and development of the grafted domain. For this study, we used several positional markers, the expression of which in normal chick (and quail, not shown) embryos will first be described below briefly, both at the stage used for grafting 3384 L. Bally-Cuif and M. Wassef Wnt-1 in met/mesencephalic grafts 3385 Fig. 3. Localization of Wnt-1 transcripts on whole-mounts HH7 (A), HH9 (B), HH11 (C, dorsal view; D, lateral view), HH12-13 (E), HH14-15 (E3) (F (lateral view),G,H (dorsal views)), E6 (K, lateral internal view of the neural tube cut along the midline) chick embryos, or E9.5 mouse embryo (I), and on a HH14 quail embryo homotopically grafted at HH10 with chick mesencephalon (Fig. 2A) and hybridized with the Ch.Wnt-1 probe (J, dorsal view). Except in A and B, where entire embryos are shown, hybridizations were performed on dissected neural tubes. In A-F, I and K, anterior is to the right. In the chick embryo, expression appears on the anterior neural folds at the level of neural tube closure (A) and scattered cells then appear laterally (hardly visible in B, arrows). Later on, expression covers the mesencephalic vesicle, and is more concentrated in a ring in its caudal part (arrowhead in C-E) and in rhombomere 4 (open arrow in C-E). A higher magnification of expression at HH11 is presented in Fig. 4G. Transient expression is visible in diencephalon at HH12-13 (arrow in E). Between E3 and E8, two delimited domains of expression are seen: the metmesencephalic ring (arrowheads in F (where the left side of the ring is visible by transparency), G,K) and the dorsal midline (arrows in F,G,K). Expression in the latter domain appears as two parallel rows of stained cells, and decreases between mes- and diencephalon (arrow in H). No expression is seen on ventral midline cells, contrary to the mouse embryo (small arrow in I). In J, the location of the graft is delimited by the doted line. Note that Ch.Wnt-1 expression is confined to the dorsal midline (arrow) and that no positive cells are found at the level of the met-mesencephalic ring. Fig. 4. Expression patterns of Wnt-1, En-2, Pax 6 and Cotx2 in the anterior chick neural tube at stages HH10-11 (A,C,E,G,H) and HH16 (B,D,F,I). The En-2 protein was detected using the mAb 4D9 (Patel et al., 1989) and whole-mount immunocytochemistry (brown in A-D), and the other markers (Wnt-1 (A,B,G), Pax 6 (C,D) and Cotx2 (E,F)) were studied by whole-mount ISH (blue in A-G). H and I are flat-mounted met-mesencephalic regions of embryonic neural tubes where Wnt-1 and Cotx2 transcripts were revealed by double-labeling ISH (in H, Wnt-1 expression is brown and Cotx2 red; the reverse is used in I). En-2 is expressed over the whole met-mesencephalic domain, and the location of theWnt-1-positive ring (arrowheads in A and B) corresponds to the region of highest En-2 expression. Pax 6 transcription occurs in the alar plate of tel- and diencephalon, and in the basal plate of rhombencephalon starting at the rhombomere 1/2 boundary. The Pax-6-negative region is delimited by the arrows in C,D. Expression excludes the En-2-positive domain. Cotx2 is transcribed in telencephalon, diencephalon and mesencephalon, and expression abruptly stops at the caudal end of the mesencephalic vesicle (arrowheads in E and F). Later expression in a remnant of the tela choroidea is indicated (arrow in F). At stage HH10-11, Wnt-1 expression is uneven, and patches of positive cells are scattered in the rostral part of the metencephalic vesicle (arrows in G). These cells (bracket in H) are Cotx2 negative. The caudal limits of Wnt1 and Cotx2 expression later become closer to one another, and are almost coincident at HH16 (I). 3386 L. Bally-Cuif and M. Wassef (HH10-11) and at the stage when grafted embryos were analyzed (after E3). Expression of Wnt-1 in the normal chick neural tube in relation to other positional markers In the following, we will designate as ‘mesencephalic’ the portions of tissue originating from the mesencephalic vesicle, and as ‘metencephalic’ those originating from the metencephalic (first rhombencephalic) vesicle. ‘Met-mesencephalic’ grafts overlap the constriction that separates these two vesicles. This nomenclature therefore only refers to morphological landmarks, and is not related to fate map (see discussion). The following morphological criteria and positional markers were used to characterize specific regions of the anterior neural tube. Expression of En-2 is a marker of the metmesencephalic domain We used the 4D9 monoclonal antibody (Patel et al., 1989) to localize En-2-positive cells on whole-mount embryos. At the stage of grafting (HH11), and as already described in previous reports (Gardner et al., 1988; Patel et al., 1989; Martinez and Alvarado-Mallart, 1990; Martinez et al., 1991; Storey et al., 1992), this antibody specifically stains the mesencephalon and metencephalic vesicles (Fig. 4A). Maximal intensity of expression is found at the junction between these vesicles, and staining progressively decreases as one moves from this region, both posteriorly into the cerebellar plate and anteriorly into the mesencephalon. As in the mouse (Bally-Cuif et al., 1992), we found that the met-mesencephalic Wnt-1-positive ring was located within the En-2-positive domain, coinciding with the region of highest En-2 expression (see Fig. 4A). This expression pattern and the relationship between Wnt-1 and En2 expressions are maintained at later stages (Fig. 4B). Expression of Pax 6 is a marker of the diencephalon and telencephalic territories To identify a diencephalic or telencephalic phenotype, we used a quail Pax 6 probe, Pax-QNR (Martin et al., 1992). This probe was found to cross-react with the chick Pax 6 mRNAs in our experimental conditions, and gave the same expression pattern in both species (see Fig. 4C,D for chick embryos): at stage HH11 (Fig. 4C) and later (Fig. 4D), Pax 6 transcripts are detected in the alar plate of the presumptive diencephalon and telencephalic vesicles, as well as in the basal plate of spinal cord and rhombencephalon with a rostral limit at the rhombomere 1/2 border. These results are in agreement with previous reports in the chick (Li et al., 1994), zebrafish (Püschel et al., 1992) and mouse (Walther and Gruss, 1991) embryos. Double staining for both Pax 6 and En-2 on the same embryos indicate that the two genes are expressed in mutually exclusive domains (Fig. 4C,D), as reported for zebrafish (Püschel et al., 1992), with gaps expressing neither gene in the rostral mesencephalon and caudal metencephalon, due to the rostrally and caudally decreasing expression of En-2. Expression of chick Otx2 (Cotx2) is a marker of the tel-, di- and mesencephalic territories The chick Otx2 gene, like its mouse homolog (Simeone et al., 1992, 1993), is expressed in the anterior neural plate since the beginning of neurulation (Bally-Cuif et al., in press). Its caudal limit of expression, initially fuzzy, becomes more sharply defined after approximately stage HH10, running through the caudal portion of the mesencephalic vesicle (see Fig. 4E,F), within the Wnt-1-positive domain, as revealed using doublelabeling whole-mount in situ hybridization (see Fig. 4G,H). Once Wnt-1 expression becomes restricted to a narrow metmesencephalic ring, the Wnt-1-positive territory extends for only one or two rows of cells further caudally (stage HH13, Fig. 4I). The cerebellar plate is Cotx2-negative. Expression is also detected after this stage in the tela choroidea (Fig. 4F, arrow), and in rhombencephalic cells bordering the tela. We therefore used Cotx2 expression as a marker of tel-, di- and mesencephalic territories, as well as of choroid plexus, versus metencephalic territories. In our experimental conditions, the Cotx2 probe cross-reacted with the quail otx2 transcripts, and the same expression pattern was observed in quail and chick embryos at equivalent stages (not shown). After an ectopic met-mesencephalic graft, Wnt-1 expression is reorganized in concert between the grafted tissue and the surrounding host In previous experiments (Bally-Cuif et al., 1992), we observed that Wnt-1 expression was maintained in grafts taken from the Wnt-1-positive region of a mouse met-mesencephalon and placed in the diencephalon of chick hosts. In order to study whether Wnt-1 expression was reorganized within the graft, and whether host Wnt-1-expressing cells were also participating in the ectopic development of the grafted tissue, we analyzed Wnt-1 expression in chimeras where various portions of quail met-mesencephalon were allowed to develop ectopically in the anterior neural tube of a chick host (Fig. 2B). For this study, we used the Ch.Wnt-1 and Q.Wnt-1 probes, which do not cross-react in our experimental conditions, so that host and graft Wnt-1 transcripts can be separately identified in the chimeras. Chick hosts grafted at stage HH10-11 with regions A (‘mesencephalic’ grafts), B (‘metencephalic’ grafts) or C (‘met-mesencephalic’ grafts) (Fig. 2B) were first analyzed after 2 days for Ch.Wnt-1 expression. In all cases, chick Wnt-1-expressing cells were found in contact with the graft (n=approx. 100). Surprisingly, in most cases (approx. 90%), the Wnt-1-expressing host cells formed a continuous line connecting the Wnt-1positive dorsal midline with the graft (Fig. 5A, open arrow). Wnt-1 expression on the midline thus seemed to extend anteriorly and to deviate on the grafted side compared to the contralateral side. In rare cases only (approx. 10%), these ectopic Wnt-1-positive cells had no connection with Wnt-1-positive dorsal midline cells (Fig. 5B). No Ch.Wnt-1-positive cells were ever observed near a portion of telencephalon or diencephalon grafted in the same location (n=12, Fig. 5J). Q.Wnt-1 was expressed in the grafted tissue in almost all embryos analyzed (98%, n=88). When both Ch.Wnt-1 and Q.Wnt-1 were analyzed using two-color whole-mount in situ hybridization (n=88), the quail and chick Wnt-1-expressing regions were always found to contact each other (Fig. 5C-I). The Q.Wnt-1-positive cells were arranged in highly regionalized patterns which depended on the type of graft. (i) In 85% of mesencephalic (type A) grafts (n=21), Q.Wnt1 was found concentrated on one side of the graft, directly in contact with the host tissue (see Fig. 5C-G). In 10% of cases, no Q.Wnt-1-expressing cells were seen, and a Ch.Wnt-1- Wnt-1 in met/mesencephalic grafts 3387 positive line was partly surrounding the graft. In the remaining 5% of cases, Q.Wnt-1 expression was very low and could not be precisely mapped. The reorganization of Q.Wnt-1 expression was never found in non-integrated grafts. The chick and quail Wnt-1-expressing zones, whether they were continuous (71% of cases, see Figs 5C; 6B) or adjacent to each other (29% of cases, see Fig. 6A), always formed a narrow domain separating the graft from the host. (ii) In 100% of metencephalic (type B) grafts (n=36), Q.Wnt1 was regionalized within the grafted tissue as a more or less complete circle-shaped positive region, dorsally in contact with the host and surrounding a tissue of thin teloid appearance (Figs 5H,I; 6C,D). Based on Wnt-1 expression in met-mesencephalon at the time of grafting (see Figs 3C,D; 4G), this expression of Wnt-1 probably results from de novo initiation of Wnt-1 transcription within the graft. Importantly, this specific spatial organization of Wnt-1 expression was also observed in non-integrated grafts (3% of cases), suggesting that it could occur independently of the surrounding host. When the graft was integrated, the chick host was found to participate in the formation of a complete Wnt-1-positive circle, either through a cluster of strongly Ch.Wnt-1-positive cells on the dorsal aspect of the circle (33% of cases) (Fig. 5I), or through a longer line of positive cells surrounding part of the tela (67% of cases) (Fig. 5H, 6C,D), usually caudally. In both cases, chick and quail Wnt-1-expressing cells were in continuity with each other. (iii) Met-mesencephalic (type C) grafts (n=31) gave more variable results concerning Wnt-1 expression, and will be detailed below, in relation with the localization of other phenotypic markers. Briefly, the two aspects described above for mesencephalic and metencephalic grafts were obtained, in various proportions depending on the exact region grafted. (iv) When portions of diencephalon or telencephalon were grafted into the same location (n=12), Wnt-1 expression was only observed in the graft in one case, as scattered clusters of positive cells, that is with no sign of reorganization (not shown). All the other grafts (i.e. 92% of cases) were devoid of Wnt-1-expressing cells (Fig. 5J). In summary, in a large majority of cases, the host and the grafted portion of met-mesencephalon were observed to cooperate to reconstruct a continuous line of Wnt-1-expressing cells delimiting territories within and around the grafted region. Identification of the territories in contact with Wnt-1positive cells during the ectopic development of met-mesencephalic regions Grafts of met-mesencephalon into chick diencephalon are able to modify the phenotype of the surrounding host tissue (Gardner and Barald, 1991; Martinez et al., 1991; Bally-Cuif et al., 1992). To define the phenotype of both the grafted and host structures in contact with the Wnt-1-positive cells, we used the Cotx2, Pax 6 and En-2 markers. The most frequent configurations are schematized in Fig. 8, and detailed observations are presented below. Mesencephalic (type A) grafts (see Fig. 6A-B′) The grafted tissue was in all cases En-2- (n=11) and Cotx2(n=4) positive, but Pax 6-negative (n=14), indicating that it had maintained its original mesencephalic phenotype. Cotx2 expression was not perturbed in the grafted region (not shown). En-2 inductions were observed in the host territory in contact with the graft in 65% of cases (n=11) (see Fig. 6A,B), principally caudal to the graft. Pax 6 transcription was generally shut off in the induced region (asterisk in Fig. 6A,B) and replaced by En-2 expression, although regions of overlap between high expression of the two genes were sometimes observed rostrally (see Fig. 6B, double arrows). In most (80%) of the cases showing En-2 induction, the quail/chick Wnt-1-positive line reconstructed in the periphery of the graft ran through the En2-positive territory, separating the graft from the induced host tissue (see Fig. 6A,B). Wnt-1-positive cells were rarely found between En-2- and Pax 6-expressing domains laterally (20% of cases). However, the ‘deflected’ Wnt-1-positive dorsal midline stripe joining the graft was found to border the induced tissue dorsally and to separate it from the contralateral Pax 6expressing cells in 50% of cases (open arrows in Fig. 6B). Metencephalic (type B) grafts (see Fig. 6C-D′) The grafted tissue was in most cases (71%, n=7) En-2 positive, although at a rather low level. No induction of En-2 in the host was ever observed around the graft. Pax 6 expression was in most cases maintained near the graft and extended caudally to the same limit as on the contralateral side (not shown). In all cases studied (n=36), the dorsally located tela-like structure bordered by Wnt-1-positive cells (stars in Fig. 6C,D) showed faint Cotx2 expression (only visible in Fig. 6D), whereas the rest of the graft appeared entirely Cotx2-negative. These two structures can therefore be identified as tela choroidea and metencephalon, respectively. In 92% of cases, Ch.Wnt-1 expression was only found to border the graft along the tela choroidea, when the latter was in contact with the host. It was rapidly interrupted as it reached Q.Wnt-1 expression and the metencephalic tissue. The latter was therefore only rarely (8% of cases) separated from Pax 6 expression by Wnt-1-positive cells, whereas the tela choroidea was in all cases surrounded by a combination of chick and quail Wnt-1-expressing cells. Met-mesencephalic (type C) grafts (Fig. 7) These grafts overlapped the constriction and were further subdivided in three categories depending on their rostrocaudal location: as shown in Fig. 2B, C1, C2 and C3 grafts contained approximately the same amount of tissue but a decreasing proportion of metencephalic territory. Grafts of type C1 (n=10), which overlap the met-mesencephalic constriction but carry a large portion of metencephalon, behaved exactly as the grafts B previously described, that is developed as a small metencephalic territory (Cotx-2−) and tela choroidea (Fig. 7A). However, En-2 expression in the graft was generally stronger and was induced in the surrounding host in 40% of cases. In these cases, Ch.Wnt-1 expression was still found to border mainly the tela choroidea, although it could surround the whole graft in some cases (Fig. 7A, small arrows). This time Ch.Wnt-1-positive cells were found in a En2-positive environment. Grafts of type C 2 and C3 (n=21) (Fig. 7B-D′) gave the same results. They formed in 95% of cases two different structures, separated by a small constriction within the grafted tissue. In all cases, one of these structures was Cotx2-positive and the other Cotx2-negative; their phenotype can therefore be identified as mesencephalic and metencephalic, respectively. 3388 L. Bally-Cuif and M. Wassef Fig. 5. Expression of Ch.Wnt-1 and Q.Wnt-1 in chick embryos bearing heterotopic (Fig. 2B) grafts of quail mesencephalon (A-G), metencephalon (H,I), or diencephalon (J). All the whole-mount preparations (A,B,I) are lateral views of dissected neural tubes, anterior to the right, and C,H are schematic drawings of such whole-mount neural tubes, with the same orientation. In A and B, Ch.Wnt-1 transcripts (blue) and the En-2 protein (brown) are detected. The grafts are En-2-positive and En-2 inductions are observed in the hosts (arrows). The Wnt-1positive dorsal midline (large arrow) and the ectopic Ch.Wnt-1-positive cells (open arrows) are indicated. The latter form in most cases a continuous line joining the dorsal midline and the graft (A). In C-J, Ch.Wnt-1 (red, arrowheads) and Q.Wnt-1 (blue, open arrows) transcripts were detected by double-labeling whole-mount ISH. The grafts in D and J are surrounded by dotted lines. C is a schematic drawing of the neural tube of the embryo flat-mounted in D, the graft is surrounded by the black line. In D, anterior is to the top, and the dorsal midline is indicated by the white dotted line. E is the corresponding section at the level indicated (bar in D), anterior to the top, counterstained with propidium iodide (F,G) to visualise quail nuclei (arrows in G). The host/graft limit is indicated by the dotted arrow in E, the quail is to the right and the chick to the left. The Ch. and Q.Wnt-1-positive domains form a continuous line (C,D), and Q.Wnt-1 transcripts are confined to the host/graft junction (E). H is a schematic drawing of the results obtained with metencephalic grafts, and I shows one example. A ring of Q.Wnt1-expressing cells (open arrow) surrounds a tela (not visible in I, but see Fig. 6C,D), and is connected with Ch.Wnt-1-positive ectopic cells (arrowhead) joining the host dorsal midline to the graft (not visible in I, but see Fig. 6C, D). J is a flat-mount of the grafted region in the case of a diencephalic graft, anterior to the top. The dorsal midline is Ch.Wnt-1-positive (arrowhead). Note that the grafted tissue is Q.Wnt-1-negative, and that no ectopic Ch.Wnt-1-positive cells are observed in contact with it. Wnt-1 in met/mesencephalic grafts 3389 Fig. 6. Expression of Wnt-1 in and around mesencephalic grafts (type A) (A-B′) and metencephalic grafts (type B) (C-D′), in relation with other positional markers. All pictures are flat-mounts of the grafted region in grafted embryos analyzed at HH16. In A and D, anterior is to the top; in B and C, it is to the right. In each case, several markers were analyzed and interpretations of the results are presented in the drawings below each figure (see key). The graft is indicated by the continuous black line, and the cuts made for flat-mounting the embryos are schematized by the irregular broken line. En-2 was detected by immunocytochemistry (brown), and all the other markers by whole-mount ISH (blue and/or red). D is a double colour ISH; Ch. and Q.Wnt-1 are red, Cotx2 is purple. When visible, the location of the dorsal midline is indicated by the small arrow on the left (A) or bottom (B) of the drawings. En-2 is induced in contact with the graft in A and B, and a Pax-6-negative region (asterisk) is found adjacent to the graft, in a faintly En-2-positive region. Dorsally, the Wnt-1-positive line often separates an En-2-positive from a Pax-6positive territory (regions indicated by the arrowheads in B). The induced En-2positive cells however often stradle the Wnt-1 line anteriorly (arrows in A and B) and laterally (when present); note, in B, that some of these cells can express both En-2 and Pax 6 (double arrow). Metencephalic grafts (C,D) develop a faintly Cotx2-positive (see D) tela-like structure (stars in C and D), easily visible in C which is a dark-field view. The tela is surrounded by a combination of Ch. and Q.Wnt-1-expressing cells, but the metencephalon (mt) is generally not. Cotx2 (D) and Pax 6 (C, visible in brightfield) expressions are not modified around the grafts. However, in contrast with grafts B and C1, a tela choroidea developed in the metencephalic part of the graft in only 14% of cases (Fig. 7B). The final anteroposterior (AP) orientation observed within the grafted tissue was strictly dependent on its AP orientation in the donor: in non-inverted grafts (Fig. 7B,D), metencephalon developed posteriorly and mesencephalon anteriorly, and the reverse was obtained in inverted grafts (Fig. 7C). In all cases, both parts of the graft strongly expressed En2, and En-2 was induced in the host, generally caudal to the graft and in contact with both its metencephalic and mesen- cephalic parts. In this type of grafts, chick and quail Wnt-1 behaved differently. In 95% of cases (n=16), Q.Wnt-1expression was found regionalized within the grafted tissue at the junction between the metencephalon and mesencephalic aspects of the graft, on the Otx-2-positive side, therefore mimicking the normal met-mesencephalic pattern (see Fig. 7C,D). Ch.Wnt-1-positive cells, in contrast, surrounded part of the graft, either its metencephalic (75% of cases) (Fig. 7B,D) or mesencephalic (25% of cases) (Fig. 7C) component. This choice seemed independent of the AP orientation of the graft. 3390 L. Bally-Cuif and M. Wassef Similarly to what was observed in type A grafts, Ch.Wnt-1positive cells were located inside the En-2-positive domain (grafted and induced), separating the graft from the host rather than two territories expressing different markers. Again, the En-2-positive induced territory was separated from the contralateral Pax 6-positive domain by the ‘deflected’ Wnt-1positive dorsal midline (not visible in the cases presented in Fig. 7 since the grafts are located very close to the midline). DISCUSSION Wnt-1 expression in the chick embryonic neural tube in relation with other markers and metmesencephalic fate map We have used PCR amplification and degenerate oligonucleotides to isolate a chick Wnt-1 cDNA. Several Wnt-1-related genes have been isolated in vertebrates (see McMahon, 1992, for a review) and encode a family of structurally related proteins. Our cDNA is much more homologous to mouse, Xenopus and zebrafish Wnt-1 than the various Wnt genes are to one another (<60%), and the expression pattern observed on the dorsal midline and met-mesencephalic region of the chick neural tube was very similar to that reported for Wnt-1 in other vertebrate species (Wilkinson et al., 1987; Molven et al., 1991; Noordermeer et al., 1989), showing that we have indeed isolated part of the chicken homolog of Wnt-1. A surprising feature was the absence of Wnt-1 transcripts on ventral midline cells of mesencephalon and diencephalon, a region which, in the mouse embryo, shows strong expression. This domain also seems to be missing in zebrafish (see Figs 6, 7 in Molven et al., 1991), indicating that it may either lack a relevant developmental function, or play an evolutionary recent, mammalianspecific, role. The setting up of Wnt-1 expression in the met-mesencephalic region appears to be a highly dynamic process, as previously noted in the mouse (Bally-Cuif et al., 1992; McMahon et al., 1992; Parr et al., 1993). A wide (although not uniform) distribution of transcripts throughout the mesencephalic vesicle at early stages (HH10-12) is rapidly turned into a narrow met-mesencephalic ring, which is maintained later on. Our homotopic quail/chick grafts (Figs 2A, 3J) indicate that this transition most probably results from turning off the Wnt1 gene anteriorly, rather than from a caudal migration of the mesencephalic Wnt-1-positive cells. The occurrence of a late, spatially restricted, phase of Wnt1 expression at the met-mesencephalic junction suggests that Wnt-1 might play a role in providing positional cues in this location, after the met-mesencephalic domain has been specified as a whole. Similarly, after a phase of broad expression involved in the specification of the wing disc (Sharma and Chopra, 1976; Couso et al., 1993), wingless expression becomes restricted to the dorsoventral boundary of the disc, where it plays a role in patterning the wing margin (Phillips and Whittle, 1993; Williams et al., 1993). In met-mesencephalon, the location of the future met-mesencephalic ring is clearly visible from stage HH10-11 onwards as a strongly Wnt-1-positive region in the caudal part of the mesencephalic vesicle. The rostral border of this Wnt-1-expressing domain seems to correspond to the rostral limit of the territory participating in cerebellar development, as defined using quail/chick chimeras (Martinez and Alvarado-Mallart, 1989; Hallonet et al., 1990); however, at this stage, this region also contributes to tectal structures (Martinez and Alvarado-Mallart, 1989; Hallonet et al., 1990). The avian met-mesencephalic fate map is not directly known at later stages. However, Itasaki et al. (1991) showed that the local influences, which, at HH10-11, have the capacity to modify the fate of rostral mesencephalon grafted in the caudalmost part of the mesencephalic vesicle, are no longer operative after HH16. This suggests that the presumptive cerebellar and tectal territories are more clearly determined and separated after HH16. The refinement of the strongly positive Wnt-1 domain thus seems to parallel that of the met-mesencephalic fate map. However, whether the location of the Wnt-1-positive ring at HH16 corresponds to the limit between the presumptive cerebellar and tectal territories cannot be ascertained. The same conclusions hold for the location of the caudal limit of Cotx2 expression, and we accordingly called ‘mesencephalic’ and ‘metencephalic’ the Cotx2-positive and -negative domains, respectively, by reference to morphology only. However, the progressive alignment of the caudal limit of Cotx2 expression with the Wnt1 ring, together with the observation that this limit is perturbed in swaying (Wnt-1−) mouse mutants (Bally-Cuif et al., unpublished data), strongly suggest that Wnt-1 expression is implicated in the positioning or the maintenance of this Otx-2+/− subdivision within the neural tube. Fate of the grafted tissue, expression of Wnt-1 and correlation with the induction of En-2 A possible early role of Wnt-1 in the regulation of expression of En genes has been suggested (Bally-Cuif et al., 1992; McMahon et al., 1992). Our results in the chick embryo add support to this hypothesis. First, as noted in the mouse (BallyCuif et al., 1992; McMahon et al., 1992), Wnt-1 expression overlapped with the En-1- (not shown) and En-2-positive domains since the earliest expression of these genes. Second, En-2 induction in our grafted embryos was closely correlated with the presence in the graft of the caudal mesencephalic vesicle, which contains the region of highest Wnt-1 expression (compare grafts B and C). Together with the disappearance of En-expressing cells in Wnt-1− mice (McMahon et al., 1992), these results suggest the possibility of crossregulations between Wnt-1 and En genes, reminiscent of those observed between their Drosophila homologs wingless and engrailed (Di Nardo et al., 1988; Heemskerk et al., 1991; Cumberledge and Krasnow, 1993). In vertebrates, these interactions might be involved in the specification of the met-mesencephalic domain. However, Wnt-1 was also expressed at the host-graft junction, near the ectopic tela choroidea, and in the ‘deviated’ dorsal midline cells, in the case of metencephalic grafts (B), which did not induce En-2. This observation suggests that Wnt1, if implicated, is not the only factor responsible for En-2 induction, since even in a permissive environment Wnt-1expressing cells of the met-mesencephalic junction and those of the dorsal midline and lining the tela choroidea do not have the same inducing capacity. However, it remains possible that the latter expression of Wnt-1 is initiated too late to influence the diencephalic phenotype of the host. Indeed, Wnt-1 expression is turned on between the cerebellar edge and the tela choroidea at a rather late stage in situ, compared to its initiation at the met-mesencephalic junction. Wnt-1 in met/mesencephalic grafts 3391 The factors governing the directionality of the En-2 inductions remain elusive at present. As already reported (Martinez et al., 1991; Gardner and Barald, 1992), although a few En-2positive cells were often found anterior to met-mesencephalic grafts, En-2 expression was mostly induced caudally and with a rostrocaudally decreasing gradient. From these previous reports, however, it could not be concluded whether or not the directionality of En-2 induction was due to the AP orientation of the graft, since in the previous reports either this orientation was not precisely determined (Gardner and Barald, 1992) or the grafts were themselves rostrocaudally inverted (Martinez et al., 1991). It is known that rostrally directed inductions can be produced when rostrocaudally inverted mesencephalic vesicles are grafted into mesencephalon (Marin and Puelles, 1994), and that En-2-inhibitory factors are present near the mes/diencephalic junction (Itasaki et al., 1991), suggesting that environmental influences might be important to restrict the extent of induction in the anterior neural tube. The use of positional markers in our study allows us to conclude (1) that a graft straddling the met-mesencephalic constriction and placed in diencephalon always maintains its intrinsic rostrocaudal polarity and (2) that the directionality of En-2 induction does not depend on the AP orientation of the graft, but rather on surrounding polarizing influences. The fate of the caudal mesencephalon varied depending on which met-mesencephalic domain it was grafted with: it developed a metencephalic phenotype when associated with caudal regions (case C1) and a mesencephalic phenotype when associated with rostral regions (case A). This indicates that this region of the neural tube is at least bipotent, and that the choice governing its fate depends on rostral and caudal influences from the surrounding tissues. This is in agreement with the results obtained by Martinez and Alvarado-Mallart (1990) and Alvarado-Mallart et al. (1990), showing that rostrocaudally inverted mesencephalic grafts can regulate their fate according to the A/P orientation of the environment. Wnt-1 expression is induced or maintained ectopically in the host in contact with a grafted portion of met-mesencephalon Our results indicate that ectopic Wnt-1-expressing host cells are always found in contact with a grafted portion of met-mesencephalon. Whether this results from a de novo induction of Wnt-1 in contact with the graft, or from the stabilization of the transient diencephalic Wnt-1 expression near the graft, is not known. The fact that these Wnt-1-expressing host cells are very rarely found in isolation, but are in most cases connected with dorsal midline cells through a continuous positive line, is puzzling. Diencephalic or telencephalic grafts placed in the same location do not have the same effect, demonstrating that this line is not due to mechanical distortion of the dorsal midline during grafting. Moreover, experiments where the grafted tissue was placed very laterally in the diencephalon, without cutting the dorsal midline, showed the same midline ‘deviation’ (not shown). Met-mesencephalic grafts are known to modify the surrounding host. It is therefore possible that they create in the host a new morphogenetic field within the diencephalic domain, and that Wnt-1 expression is induced as a result of discontinuities in positional values. The midline of the neural tube might dorsally limit the field of influence of the grafts. A continuous region of discontinuity in positional values, joining the dorsal midline and the graft, might therefore be created in the host, and Wnt-1 expression induced in this location. The exact ‘phenotype’ of the ectopic Wnt-1-positive cells is not clear: because their presence is not correlated with En-2 induction, it appears more likely that they have dorsal midline properties, rather than met-mesencephalic characteristics. However, as will be discussed below, they are sometimes positioned as a border between territories of different phenotypes, which is reminiscent of the met-mesencephalic Wnt-1expressing cells. It would be interesting to compare the location of these cells with that of known dorsal midline markers. Wnt-1 expression is reorganized in concert between the host and the graft to border specific metmesencephalic domains The spatial pattern of Wnt-1 expression within the grafted tissue corresponded precisely to the origin and fate of the graft: around the tela choroidea for metencephalic grafts, between the metencephalon and mesencephalic regions for met-mesencephalic grafts, and on the edge of the grafted tissue for mesencephalic grafts. A constant feature, however, was the participation of host Wnt-1-expressing cells to complete or reinforce this pattern, so that a continuous Wnt-1-positive line was formed. We have recently observed in the swaying Wnt-1− mouse mutants that Wnt-1 expression was necessary boundary formation between mesencephalon and metencephalon, and between metencephalon and choroid plexus (Bally-Cuif et al., unpublished data). In the case of our grafting experiments, the ectopically organized Wnt-1 line might also mark the location of a boundary between differently developing territories. The observation, further discussed below, that it is frequently found between regions expressing different markers, argues in favor of this interpretation. In the case of mesencephalic or met-mesencephalic grafts, the ‘deviated’ Wnt-1-positive dorsal midline was frequently found to separate the grafted and induced En-2-positive territory from the contralateral Pax 6-positive domain. In some cases, the anterior host/graft junction almost corresponded to the limit between En-2 and Pax 6 expressions; but a few En2-positive cells were generally found to straddle the Wnt-1 line. Interestingly, En-2 and Pax 6 were generally coexpressed in these cells. By analogy with engrailed and cubitus interruptusD expressions at the AP boundary of the Drosophila wing disc (Blair, 1992), it is therefore possible that these En-2-positive cells do not necessarily correlate with the future development of a met-mesencephalic phenotype, and that the Wnt-1 line indeed separates two domains that will evolve as different structures. The study of grafted embryos at later stages would help to resolve this point. In the case of metencephalic grafts, the Wnt-1-positive line was always found to separate Cotx-2positive and -negative territories. These observations, in particular in the case of metencephalic grafts, indicate that Wnt-1 expression can be ectopically turned on at the junction between territories of different specification. This is strongly reminiscent of the Drosophila wing imaginal disc system, where ventral-type cells become surrounded by margin structures when they are artificially induced within the presumptive dorsal territory of the disc (Diaz-Benjumea and Cohen, 1993). Whether this expression of Wnt-1 plays some active role in the development of the territories artificially placed in contact 3392 L. Bally-Cuif and M. Wassef cannot be directly concluded from our experiments. Several expression might be more generally induced at the junction arguments, based on the study of Wnt-1− mutant mice, however between rostral metencephalon or choroid plexus and a region suggest that this is the case, at least for metencephalic grafts. of different specification, and be involved in stabilizing this First, in the Wnt-1neo mutants, the metencephalic tissue starts metencephalic domain. This proposal would be in keeping with to degenerate when it gets in contact with rostral mesenthe general mechanism proposed by Meinhardt (1983), who cephalic or diencephalic regions (McMahon et al., 1992), after the caudal mesencephalon has been deleted. This observation suggests that some stabilizing factors, normally present in the caudal mesencephalon, are required to allow cerebellar development, factors that cannot be restored in the absence of Wnt-1. It is therefore tempting to speculate that the Wnt-1 protein itself might be the factor exerting this stabilizing function, in situ and more generally in the case of our ectopic metencephalic grafts. Second, the metencephalic domains that induce a Wnt-1 line in their contact when ectopically grafted are those that are preferentially affected in the Wnt-1− mutants (see McMahon et al., 1990; Thomas and Capecchi, 1990), being either deleted (rostral cerebellum) or abnormally patterned (edge of the tela choroidea) (Bally-Cuif et al., unpublished data). For example, in type B grafts, Wnt-1expressing cells were found to surround the tela choroidea, but not the cerebellar tissue, and it is known that the lateral and caudalmost parts of the cerebellum can be maintained in some Wnt-1− mutants (Thomas and Capecchi, 1990; Thomas et al., 1991) whereas the edge of the tela is always abnormal (BallyCuif et al., unpublished data). Also, when portions of presumptive rostral cerebellum are grafted together with the metencephalic vesicle (type C1), Wnt-1 expression can be found to surround part of the metencephalic tissue and not only the tela (see Fig. 7A). Likewise, rostral cerebellum always disappears in the Wnt-1− mutants. Taken together, these observations suggest that the induction of Wnt-1 expression plays an active role in the development of our grafts, possibly by providing them with Fig. 7. Expression of Wnt-1 in and around met-mesencephalic grafts of type C1 (A,A′) and stabilizing cues. C2/C3 (B-D′), in relation with other positional markers. The symbols used and the key are the same as in Fig. 6. Grafts of type C1 behave identically to metencephalic grafts, but inductions In the normal embryo, Wnt-1 is of En-2 can be observed (arrow in A). Ch.Wnt-1-expression bordering the tela and, in this expressed between metencephalon and case, most of the graft, is indicated (small arrows); these cells are within the En-2-positive mesencephalon, and between metendomain. Grafts of type C2/C3 can be divided into a Cotx2-positive (mesencephalon, ms) and a cephalon and choroid plexus, and our Cotx2-negative (metencephalon, mt) part, separated by Q.Wnt-1-expressing cells (see C,D). study of the swaying mice (Bally-Cuif In C, but not in B and D, the graft was placed in the host with a rostrocaudally inverted et al., unpublished data) indicates that it orientation; note that this orientation has been maintained. In rare cases, choroid plexus is is implicated in maintaining the spatial formed (star in B). En-2 expression is induced in all cases around the graft (arrows in B-D), integrity of these domains. From the sometimes straddling the Wnt-1-positive line anteriorly (double arrow in B). In D, the brown results presented in this paper, we line within the metencephalic domain is a fold of the tissue, which was flat-mounted in would like to propose that Wnt-1 double thickness. Wnt-1 in met/mesencephalic grafts 3393 Fig. 8. Schematic representation of the results obtained with heterotopic grafts (Fig. 2B). The expression of the different markers (see key) is represented at the time of grafting (left panel) and at the time of analysis in the grafted region (right panel). The graft is surrounded by a black line. Case C2/C3 represents a rostrocaudally inverted graft. The dorsal midline is to the left, between the two Ch.Wnt-1-positive lines. suggested that the juxtaposition of differently specified territories leads to the establishment of signalling boundaries implicated in patterning the adjacent domains. We wish to thank P. Charnay and C. Goridis for their critical reading of the manuscript, E. Boncinelli for providing the c-otx2 probe, M. Hallonet for the Q.Wnt-1 probe and S. Saule for the Pax QNR probe. We are also grateful to C. Goridis and to the members of his group for providing laboratory facilities and advice for the cloning of the Ch.Wnt-1 cDNA. Finally, we are greatly indebted to B. 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