Late emigrating neural crest cells migrate specifically to the exit points of cranial branchiomotor nerves

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1 Development 122, (1996) Printed in Great Britain The Company of Biologists Limited 1996 DEV Late emigrating neural crest cells migrate specifically to the exit points of cranial branchiomotor nerves Christiane Niederländer and Andrew Lumsden Department of Developmental Neurobiology, UMDS Guy s Hospital, London SE1 9RT, UK SUMMARY Morphological segmentation of the avian hindbrain into rhombomeres is also reflected by the emergent organisation of branchiomotor nerves. In each case, the motor neurons of these nerves lie in two adjacent rhombomeres (e.g. of the Vth nerve in r2 and r3, VIIth in r4 and r5 etc.), and their outgrowing axons emerge into the periphery through defined exit points in rhombomeres r2, r4 and r6, respectively. Sensory axons of the cranial ganglia also enter the neuroepithelium at the same points. Motor axon outgrowth through experimentally rotated rhombomeres has suggested that a chemoattractive mechanism, involving the exit points, may form a component of their guidance. Yet so far, nothing is known about the establishment of the exit points or the identity of the cells that form them. In this study, we describe a group of late emigrating cranial neural crest cells which populate specifically the prospective exit points. Using chimaeras in which premigratory chick neural crest had been replaced orthotopically by quail cells, a population of neural crest was found to leave the cranial neural tube from about stage 10+ onwards and to migrate directly to the prospective exit points. These cells define the exit points by stage 12+, long before either motor or sensory axons have grown through them. The entire neural crest population of exit point cells expresses the recently described cell adhesion molecule c-cad7. Further, heterotopic grafting experiments show that midbrain and spinal cord crest, grafted at late stages in place of r4 crest, share the same migratory behaviour to the facial nerve exit points and express the same markers as cells contributed by the native r4 crest. It was not possible to generate new exit points in odd numbered rhombomeres simply by experimentally increasing their (normally insignificant) amount of crest production. Initiation of the exit point region probably lies, therefore, in the neuroepithelium. Key words: rhombomeres, neural crest, branchiomotor neurons, quail-chick chimaeras, fate map, Krox-20, c-cadherin7 INTRODUCTION The mechanisms that guide outgrowing motor axons from their origin in the neuroepithelium into the periphery and then to their final target are only partly understood. Axons of spinal motor neurons of the trunk, for example, grow away from the floor plate and exit the neural tube in a continuous band along its anteroposterior axis, but at a specific dorsoventral level. Once in the periphery, axons form nerve bundles due to the instructive action of the adjacent segmented mesoderm: a chemorepulsive signal in the posterior halves of the somites directs spinal motor axons to grow toward the anterior half somites and thus to form the spinal motor nerve bundles (Keynes and Stern, 1984). Furthermore, it has been shown recently that the determination of motor neurons in the trunk to grow towards specific groups of muscles is correlated with the expression of combinations of LIM-homeodomain transcription factors in subgroups of spinal motor neurons (Tsuchida et al., 1994). In the head, the segmentation of the neuroepithelium of the hindbrain (Vaage, 1969; Lumsden, 1990) coordinates the development of the cranial motor nerves. In particular the branchiomotor nerves reflect this rhombomeric organisation of the hindbrain (Lumsden and Keynes, 1989). Cell bodies of motor neurons that make up the trigeminal nerve lie in rhombomere (r) 2 and r3, while the facial neurons are located in r4 and r5 and the glossopharyngeal neurons in r6 and r7. Outgrowing branchiomotor axons grow dorsally away from the floor plate, remain for some distance within the neuroepithelium and exit into the periphery through defined points in the alar plate of r2, r4 and r6. Hence, axons from odd numbered rhombomeres must turn rostrally to reach their exit points in the adjacent rhombomere. In growing towards and through these exit points, motor axons are grouped to form bundles which emerge into the periphery. The branchiomotor nerve exit points are also the entry points for ingrowing sensory axons. It has been shown that the floor plate acts as a chemorepellent for outgrowing branchiomotor axons of the hindbrain (Guthrie and Pini, 1995). Floor plate chemorepulsion has also been shown to act on trochlear motor axons and in this case could be attributed to netrin-1 (Colamarino and Tessier- Lavigne, 1995). Other studies suggest that, once in the periphery, outgrowing motor axons have their next intermediate target in the sensory ganglia, lying adjacent to the rhombomere that contains their exit point (Moody and Heaton, 1983c; Heaton and Wayne, 1986). Thus, it appears from a number of studies that the outgrowth of motor axons in the head and in the spinal cord, from their cell bodies to their final targets

2 2368 C. Niederländer and A. Lumsden in the periphery, involves their stepwise guidance by chemorepulsive and chemoattractive landmarks along their way. The pattern of branchiomotor axon outgrowth to the nerve exit points, which results in nerve formation, suggests that the exit points represent one of the chemoattractive intermediate targets guiding motor axons. Cell surface molecules in the neuroepithelium do not seem to be required to guide motor axons to the exit points. This has been shown by experiments in which odd-numbered rhombomeres were inverted in their anteroposterior orientation: the outgrowing motor axons still turn towards their anterior lying exit points (Guthrie and Lumsden, 1992). To date, the morphology and cellular composition of the exit point region is unknown and exit points only become distinguishable once sensory and motor axons have traversed through them. It is likely that the establishment and maintenance of the exit point region involves a number of different cells. The expression of genes which mark distinct cell groups at the exit points suggests the existence of specified cells at these sites. We have been interested in the role of neural crest cells in establishing exit points for motor axons, focusing on the branchiomotor neurons of the hindbrain. Previous studies have shown that the amount of neural crest produced along the dorsal aspect of the neural tube varies greatly between different rhombomeres (Lumsden et al., 1991; Sechrist et al., 1993). Rhombomeres 2, 4 and 6 which contain the exit points, produce (together with r1) the majority of neural crest cells in the hindbrain, whereas r3 and r5 are massively depleted of crest cells by apoptosis (Graham et al., 1993, 1994). Neural crest in the hindbrain migrates in three streams lying adjacent r2, r4 and r6, whose prominence relates, at least in part, to the crest depletion of r3 and r5, and along a number of dorsoventral pathways, one of which leads crest cells along the side of the basal lamina of the neural tube (Detwiler, 1937; Noden, 1988). The predominant neural crest production of the rhombomeres which contain the branchial nerve exit points and the pathway of some of the emigrating crest cells along the side of the neural tube prompted us to examine the role of neural crest cells in determining the exit point region. This study reveals a subpopulation of neural crest cells, emigrating from the neural tube of the hindbrain only shortly before crest emigration ceases, which targets predominantly the prospective exit point region. We have analysed the time course of emigration in this cell population and some aspects of its action at the target region using quail-chick chimaeras. The results show that neural crest cells, leaving the hindbrain in r2 from stage 10+ onward and in r4 from stage 11 onward, migrate specifically to the prospective branchiomotor exit point regions, which they reach at or before stage 12+. These cells therefore arrive at exit points considerably in advance of either sensory or motor axons, which do not reach the exit points until at least stage 15 (Newgreen and Erickson, 1986; Moody and Heaton 1983a; Covell and Noden, 1989). In a parallel approach, DiI iontophoresis was used to label small groups of premigratory crest cells which were also found to migrate to the exit points. These crest cells express c-cad7 and Krox-20 (r2 and r4 only). The cells do not become part of the sensory ganglia, rather they attach closely to the surface of the neural tube, frequently bulging into the neuroepithelium through perforations in the basal lamina. Heterotopic grafts of midbrain or spinal cord neural crest into the hindbrain region show that crest cells from all axial levels can contribute to the exit points. These cells appear to be targeted to this region by a property of the neuroepithelium, which may reside in its basal lamina. The signal retaining crest cells at the exit point region seems to be universally recognised by neural crest cells, making it likely that the time point of emigration from the neural tube or the time of maturation of the exit point targets at the neuroepithelium surface play a role in determining whether neural crest cells accumulate at the exit points. MATERIALS AND METHODS Neural crest transplantation experiments Fertile hens eggs were obtained from a mixed flock (Poyndon Farm, Enfield) and incubated in a forced draft incubator at 38 C to stage Quail eggs were obtained from Rosedean Farm, Cambridgeshire and incubated to the same stages. Hens eggs were prepared for grafting as described before (Simon et al., 1995). A narrow region of the dorsal neural tube at the hindbrain level was removed using needles flame-sharpened from 100 µm pure tungsten wire. This region was replaced by the equivalent dorsal piece of neural tube from a stage matched quail embryo. For fate mapping, the grafted region was orthotopic. Further experiments used heterotopic grafts from the spinal cord and midbrain, transplanted into r4, or grafts of r4 transplanted into the r3 position. Eggs were resealed with electrical tape and incubated for a further hours. DiI iontophoresis Hens eggs were incubated to stage and prepared as for grafting. They were then placed under an epifluorescence microscope and viewed at 20 magnification using an ultra long working distance objective. An aluminosilicate glass electrode micropipette was tipfilled with DiI and backfilled with 1 M KCl solution. The electrode was held in a micromanipulator and its tip positioned over the dorsal neural tube of the chick embryo. DiI was released from the electrode for a few seconds by closing an electric circuit through the electrode and the egg albumen with a 9 Volt battery. Extracellular release of dye was thus spread onto the membrane of a small group of cells. The success of the injection was monitored by a brief exposure to fluorescence. Eggs were resealed and incubated for further 24 hours. For sectioning, embryos were embedded in 20% gelatine and fixed in 4% paraformaldehyde; they were then cut at 50 µm on a vibratome and viewed on a BioRad MRC 600 confocal microscope. Immunohistochemistry Fixed embryos were stained with the QCPN antibody (developed by Carlson) using an indirect immunoperoxidase method (Lumsden and Keynes, 1989; Guthrie and Lumsden, 1992). Stained embryos were viewed as whole mounts or embedded for wax, cryo or ultrathin sectioning. Light microscopic sections were stained with toluidine blue. The QCPN monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank of the Johns Hopkins University School of Medicine, Baltimore. Electron microscopy Embryos were fixed for 4 hours at 4 C in 2.5% glutaraldehyde in 0.1 M phosphate buffer (ph 7.3). After washing overnight in phosphate buffer, embryos were osmicated in 1% aqueous osmium tetroxide and dehydrated in an ascending methanol series. After embedding in Epon (TAAB), semithin and ultrathin sections were cut. Some sections were then stained again using lead citrate and uranyl acetate. Semithin sections (1 µm) were stained with toluidine blue and viewed under the light microscope. Ultrathin sections were viewed at 75 kv in a Hitachi H700 transmission electron microscope.

3 Cranial nerve exit point cells 2369 Fig. 1. In situ hybridisation with probes that are expressed at the branchiomotor exit point region. (A) Dorsal view of a stage (st.) 16 whole-mount embryo probed with c-cad7. The branchiomotor nerve exit points and the motor column inside the neural tube are labelled. (B) Transverse section through facial exit point region of a st. 16 embryo probed with c-cad7. (C) Coronal section through st. 15 hindbrain of Krox-20 probed embryo. Anterior is at the top. The staining adjacent to r4, the unstained rhombomere, marks the facial exit point. (picture courtesy of Dr Isobel Heyman and Dr Cairine Logan). (D) Section through the facial exit point of a Krox-20 in situ hybridised st. 17 embryo. Scale bar: A, 100 µm; B-D, 50 µm. Fig. 2. Diagram of the different grafting procedures. (A) Isotopic and isochronic dorsal tube grafts consisting of either r2-r4 or r4-r6 and iontophoretic DiI injection into the same region. (B) Heterotopic grafts replacing chick r3 dorsal tube with quail r4 dorsal tube. (C) Heterotopic grafts replacing r4 dorsal tube with midbrain or spinal cord premigratory crest. Probes for in situ hybridisation The c-cad7 probe was a kind gift from Dr S. Nakagawa and Dr Masatoshi Takeichi. The region used for the in situ hybridisation studies was the 450 bp PCR-fragment previously described (Nakagawa and Takeichi, 1995). The Krox-20 probe was a 600 bp Pst1-Apa1 fragment from the mouse gene, kindly provided by Dr David Wilkinson (Wilkinson, 1992). Whole-mount in situ hybridisation Digoxigenin-labelled riboprobes (Boehringer, UK) were used to detect gene transcripts. For Krox-20 in situs the protocol used was that of Dr David Wilkinson (Wilkinson, 1992) with the omission of the proteinase K step. For c-cad7, the protocol used was that of Domingos Henrique and David Ish-Horowicz (Henrique et al., 1995) with the omission of the proteinase K step. The protocol is as follows. Embryos were fixed in 4% paraformaldehyde, washed twice in PBT (PBS with 0.1% Tween-20), dehydrated by washing in 50% methanol/pbt and 100% methanol and then rehydrated by passing them through a methanol/pbt series. Embryos were then washed twice with in PBT, once in hybridisation buffer/pbt (1:1) and then preincubated in hybridisation buffer for 1 hour at 70 C. Embryos were transferred into hybridisation buffer containing 1 µg/ml of DIGlabelled riboprobe. The hybridisation buffer consisted of 50% formamide, 1.3 SSC, 5 mm EDTA, 50 µg/ml yeast RNA, 0.2% Tween-20, 0.5% CHAPS and 100 µg/ml heparin. After hybridisation, the embryos were washed twice at 70 C in hybridisation buffer followed by one wash at the same temperature in MABT/hybridisation buffer (1:1). MABT consisted of 100 mm maleic acid, 150 mm NaCl and 1% Tween-20, final ph 7.5. The embryos were then washed in MABT followed by washing in MABT containing 2% Boehringer Blocking reagent (Boehringer, UK) and 20% goat serum. The embryos were incubated overnight in the same solution containing alkaline phosphatase coupled anti-dig antibody (Boehringer, UK) at a 1:2000 dilution. After incubation with the antibody, the embryos were washed in MABT over the next day. At the end of the day the Fig. 3. Labelling of late emigrating neural crest in the hindbrain in quail-chick chimaeras stained with the QCPN antibody, in wholemount views (A,B) and viewed confocally in transverse sections after iontophoretic DiI injection (C,D). (A) Dorsal view of hindbrain of a st. 14 embryo with r2-r4 grafted at st. 11. Quail crest cells can be seen at the trigeminal and facial exit points. (B) Side view of a st. 14+ embryo with r4-6 grafted at st. 11+; anterior is to the right, dorsal is up. Quail crest cells are at the facial, glossopharyngeal and vagal exit points. ot, otocyst. (C) Trigeminal exit points in st. 15 embryo after injection into r2 at st. 11. (D) Facial exit point in st. 15 embryo after injection at st. 11. Scale bar: A-C, 100 µm; D, 50 µm. embryos were washed in NTMT (100 mm Tris-HCl ph 9.5, 100 mm NaCl, 50 mm MgCl 2, 1% Tween-20) twice for 10 minutes. The colour reaction was developed in NTMT containing 5-bromo-4-chloro-3- indolyl phosphate. Quail-chick chimaeras were then immunohistochemically stained using the QCPN antibody (see above). For sectioning, embryos were embedded in OCT compound, frozen and cut at 13 µm in a cryostat. RESULTS Specific gene expression marks the exit point sites Motor nerve exit points become readily distinguishable once

4 2370 C. Niederländer and A. Lumsden axons have passed into and out of the neural tube. The expression of certain genes seems to relate to future nerve exit point regions. The chick homologue of cadherin-7, c-cad7, a calciumdependent cell adhesion molecule, has been cloned recently (Nakagawa and Takeichi, 1995). The expression pattern is associated with cranial nerve development, and Fig. 1A,B shows its expression at prospective branchiomotor nerve exit points. C-cad7 is first expressed by neural crest cells leaving the midbrain at stage 10. Subsequently, between stage 10-11, neural crest cells of the hindbrain start expressing c-cad7 at the time of their emigration out of the neural tube. The c-cad7- positive signal initially can be seen in a short stream away from the hindbrain but between stage 12 and 13 the signal becomes confined to the exit points of developing cranial nerves. C-cad7 is also expressed in the trunk by crest cells at the dorsal and ventral root. Apart from neural crest, c-cad7 expression is found throughout the motor column in the neural tube (Nakagawa and Takeichi, 1995). Krox-20 is a transcription factor of the zinc finger family whose transcripts are restricted to r3 and r5 of the hindbrain (Wilkinson et al., 1989). In addition, there is a distinct domain of Krox-20 expression in the neural crest at sites which have been identified as boundary caps adjacent to r2 and r4, described both in mouse (Wilkinson et al., 1989) and in chick (C. Logan, unpublished data). In chick, this expression can be seen from about stage 14 onward; transverse sections of wholemount in situs probed for Krox-20 transcripts (Fig. 1C,D) show this boundary cap at the same site as c-cad7 expression in the exit points. These expression patterns suggest that the prospective exit point region is populated by specialised cells before the arrival of motor axons. A late emigrating population of cranial neural crest cells specifically targets nerve exit points We were interested in determining the role of neural crest in exit point formation. Because a universal and unique marker for neural crest cells has not been described, we marked neural crest cells by making quail-chick chimaeras. By replacing a very narrow region of chick dorsal neural tube with the same region of isochronic quail embryos (Fig. 2A), labelled crest cells were produced whilst ensuring that as little as possible of the remaining neural tube cells were labelled. Quail cells were visualised immunohistochemically using an antibody (QCPN) directed against a perinuclear epitope in quail cells. At around stage 10+ dorsal neural tube from r2-4 or r4-6, that is premigratory neural crest, was replaced by the same region of quail embryos. Because crest emigration in the hindbrain starts at around stage 9 and lasts for about 24 hours (Newgreen and Erickson, 1986; Lumsden et al., 1991) this procedure led to the labelling of neural crest cells that emigrate from the neural tube comparatively late. These crest cells ended up predominantly at the branchiomotor nerve exit points (Fig. 3A,B). Crest cells from r2-4 were found at the trigeminal (r2) and facial (r4) exit points (Fig. 3A) and crest cells from r4-6 migrated to the facial, glossopharyngeal (r6) and eventually the vagal (r7) nerve exit points (Fig. 3B). To confirm these results, premigratory neural crest cells were iontophoretically labelled with DiI (Fig. 2A). As in the quail-chick chimaeras, we found neural crest cells migrating specifically to the branchiomotor nerve exit points (Fig. 3C,D). The observed migration behaviour in the quail-chick chimaeras is therefore not due to the changed environment or the grafting procedure: the same migration pattern to the exit points is found in embryos where neural crest was labelled using the much less invasive method of DiI iontophoresis. Crest cells could be seen at the exit points as early as stage 12+, a stage at which neither sensory nor motor axons have yet grown through the exit points (Fig. 4A shows a section through a stage 13 embryo). This is, however, only the earliest time point included in this study, since it is difficult to combine the grafting procedure with a shorter incubation time which would not allow sufficient healing of the graft. The fact that the exit point region is already populated by many crest cells suggests that the first time these cells can be found at prospective exit points is even earlier. This view is also supported by the expression pattern of c-cad7. In embryos ranging from stage 16 to 21, the neural crest cells stayed at the exit points, remaining separate from the proximal part of the ganglia (Fig. 4B). At these later stages the crest cells were seen to bulge deeply into the side of the neural tube (Fig. 4C,D), suggesting that the basal lamina of the neural tube is no longer present there. Some of the quail-chick chimaeras were embedded for high resolution light microscopy and electron microscopy, to analyse in greater detail the interaction of crest cells with the basal surface of the neural tube. The quail cells were seen to come close to the neural tube and at the electron microscopic level to bulge into it (Fig. 5). The basal lamina of the neural tube looked ruffled at the exit point region in contrast to elsewhere. Fig. 5C,D shows labelled quail-cells in non-counterstained sections coming close to the neural tube. On a parallel section to that shown in Fig. 5D, uranyl acetate and lead citrate staining was applied to visualise the structure of the basal lamina at the exit points (Fig. 5E,F,G). We found that at the site where the labelled cell appears to bulge into the neural tube the typical double layered structure of the basal lamina is absent (compare Fig. 5F with 5G) and crest cell and neuroepithelium are in direct contact. The specificity of the prospective exit points is determined before the arrival of neural crest cells To determine whether the generation of exit points is dependent on the amount of crest cells generated at a given rhombomeric level, or on the specific anteroposterior identity of the crest cells, heterotopic grafts were made. Quail dorsal r4 was transplanted in place of dorsal r3, thereby creating a continually high emigration level of crest cells from r2, r3/r4 and r4 (Fig. 2B); r3 usually gives rise to a greatly reduced number of neural crest cells (Graham et al., 1993; Sechrist et al., 1993; Birgbauer and Fraser, 1994). In these embryos, the r4 quail cells, coming from the axial level of r3 are seen to migrate both anterior and posterior to the trigeminal and facial nerve exit points, respectively, showing no specificity for their native exit point, the facial, but also not generating an additional exit point in r3, the level of their emigration from the neural tube (Fig. 6A). This suggests that the initial cues for exit point formation lie in the neuroepithelium itself. Heterotopic grafting experiments were also performed which included premigratory neural crest of midbrain or spinal cord level grafted in place of dorsal r4 (Fig. 2C). These neural

5 Cranial nerve exit point cells 2371 crest cells do not normally form exit points for mixed sensory/motor nerves, since the latter form only at hindbrain level. However, the quail-chick chimaeras resulting from these experiments are indistinguishable from chimaeras with orthotopic grafts (Fig. 6B,C). This further strengthens the possibility that the initial clues for exit point formation lie within the neuroepithelium, and are independent of both the amount of available crest and its specific anteroposterior level of origin. Gene expression and the late neural crest cells at the exit points are congruent Our results suggested that the late emigrating neural crest cells we describe are the same cells that express Krox-20 and c-cad7 at the exit points. We have confirmed this by performing double labelling using in situ hybridisation on quail-chick chimaeras. We find the expression site of c-cad7 and the target site of late emigrating neural crest cells are coextensive (Fig. 7A). The expression pattern of Krox-20 makes it very likely that late emigrating neural crest cells at the exit point of the trigeminal and facial nerves also express Krox-20. Moreover, double labelling with c-cad7 in the heterotopically grafted embryos where midbrain or spinal cord crest was grafted into r4, shows that these cells express the appropriate marker for their new site (Fig. 7B). DISCUSSION This study is the first to describe a specific cell population that delineates the future site of the branchiomotor nerve exit point prior to its penetration by axons from inside or outside the hindbrain. The differentiation of motor neurons and the subsequent outgrowth of motor axons has been reported to occur from stage 13 onwards. The bulk of early motor axons passes through the exit points around stage 15, even though the earliest axons might reach the exit points as early as around stage (Noden, 1980; Moody and Heaton, 1983a,b,c; Covell and Noden, 1989; Guthrie and Lumsden, 1992). The population of late emigrating crest cells described here however, were found at the prospective exit point at least from stage 12+ onwards and very likely even earlier, judged by the amount of crest cells found at the exit points at the earliest stage examined. This subpopulation of crest cells is the earliest marker of the region where the exit point will form. In situ hybridisation showed that this cell population is also distinguished by its specific expression of c-cad7 and Krox-20. (Nakagawa and Takeichi, 1995; Wilkinson et al., 1989). Krox- 20 appears to be expressed only by cells at the trigeminal and facial exit points. C-cad7 mirrors the behaviour of the exit point crest cell population even more closely. C-cad7 expressing cells can be seen to emigrate from the neural tube of the hindbrain between stage This matches the results obtained in our grafting experiments. The neural crest cells adhere very closely to the neural tube and remain separate from the nearby proximal pole of the cranial sensory ganglion. At later stages, the cells are frequently seen to bulge into the neural tube and electron microscopic analysis has confirmed that the basal lamina of the neural tube is absent at these sites. Although this population of crest cells is the earliest marker of the region where the hindbrain exit point will form, the demarcation of presumptive motor axon exit sites in the spinal neural tube by migratory cells has been observed before. Lunn et al. (1987) describe a population of ventral neural tube cells that appear to breach the basal lamina from the inside of the neural tube and coalesce at the future ventral root before the arrival of motor axons. The neural crest cells we describe here are candidates for the secretion of molecules to guide motor axons over their intraepithelial course and maybe also to attract incoming sensory axons to the exit point (Guthrie and Lumsden, 1992). Removal of the exit point cells and subsequent analysis of motor axon pathfinding is an obvious test of this possible role. Although the regenerative capacity of neural crest at the hindbrain level (Scherson et al., 1993) rules out simple ablation experiments, we are undertaking experiments that combine extensive ablation of the neural crest together with the prevention of contact between the residual neural tube and overlying surface ectoderm, contact with which appears to be required for neural crest regeneration (Liem et al., 1995). Other possible candidates for the guidance of outgrowing branchiomotor axons include the specialised cells of the neuroepithelium which presumably reside at the site of the exit point and the ingrowing sensory axons from the cranial ganglion that use the motor neuron exit point as their entry point into the CNS. Equally, motor axons reaching the exit point before sensory axons could serve as guiding targets for ingrowing sensory axons. The first sensory and motor axons appear to enter and exit the brain at around the same time, between stage in the chick; however, the timing of the ingrowth of sensory axons versus outgrowth of motor axons is not known with precision. Studies aimed at determining whether the sensory or motor axons reach the exit points first have been inconclusive (Noden, 1980; Moody and Heaton, 1983a-d; Covell and Noden, 1989). Late emigrating neural crest cells are the first manifestation of the prospective exit point region, but heterotopic grafts show that these crest cells themselves do not determinate the site of the exit point. The reduced amount of crest cells and the absence of exit points in the odd numbered rhombomeres initially raised the possibility that the high level of crest cell generation in even numbered rhombomeres would elicit exit point formation in the same rhombomere, since the migration pathway of some neural crest cells extends ventrally alongside the neural tube. By transplanting dorsal r4 in place of dorsal r3, however, the amount of neural crest cell generation was experimentally raised at the r3 level and yet these embryos still failed to form exit points in r3. Moreover, r4 cells that at their normal site of origin are seen to migrate to the facial exit point in that rhombomere, migrate to both the trigeminal and facial exit points when grafted to r3, showing no preference for their normal destination. The r4 into r3 grafting experiment also points out another feature of the exit point region: whatever signal from the neural tube prompts late emigrating neural crest cells to migrate there, it is recognised by crest cells from different axial levels in the hindbrain and it is the same for all exit points. Neural crest cells seem to target the closest exit point to their migration path. Surprisingly, crest cells originally from axial levels that do not have mixed motor-sensory nerves, i.e. crest cells from the midbrain or spinal cord levels, are targeted to the branchial

6 2372 C. Niederländer and A. Lumsden Fig. 4. Transverse sections through the trigeminal exit point at different stages. (A) St. 13. (B) St. 17, the exit point cells do not spread out into the proximal part of the ganglion. g, ganglion. (C) St. 19; neural crest cells are closely attached to the neural tube, giving the impression of bulging into it. (D) Higher power view of the boxed area in C. Scale bar: A-C, 50 µm; D, 5 µm. Fig. 5. Transverse sections through quail-chick chimaeras (A,C) 1 mm semithin (A) and ultrathin (C) section through facial exit points of st. 16 chimaeras grafted at st (B,D) 1 mm semithin (B) and ultrathin section (D) through trigeminal exit point of st. 14 embryo grafted at st. 11. (C-G) Electron micrographs. E-F are from a parallel section to D, but additionally stained with lead citrate and uranyl acetate to enhance the contrast. The cells seen in D is marked with an arrow in E. (F,G) High power views of the cell s membrane adjacent to the neural tube (sites marked with arrowheads in E). In F the basal lamina on the side of the neural tube is clearly visible, whereas further along (G) where the labelled cell seems to indent into the neural tube, the basal lamina is absent. The interface between the two cells in G is directly under the basal lamina seen in F. nt, neural tube. Scale bar: A, 100 µm; B, 10 µm; C, D, 5 µm; E, 2.5 µm.

7 Cranial nerve exit point cells 2373 Fig. 6. Quail-chick chimaeras with heterotopic dorsal neural tube transplants. (A) Dorsal whole-mount view of a st. 16 embryo where the r3 tissue had been replaced by quail r4 tissue, the transplanted r4 crest cells have migrated anterior to the trigeminal exit points and posterior to their native facial exit points. (B) Dorsal whole-mount view of a st. 14 embryo where r4 had been replaced by midbrain tissue. (C) Transverse section through a st. 14 embryo where r4 dorsal tube had been replaced by spinal cord premigratory crest. Scale bar: A,B, 200 µm; C, 100 µm. nerve exit points exactly as normal when heterotopically grafted. These ectopic cells also express the appropriate markers for their new environment. It is therefore likely that proximity to the prospective exit points and the time of emigration from the neural tube are the factors which determine the migration target of these cells. Our experiments thus show that the determinant of the exit point site must lie in the neural epithelium itself. Anteroposterior patterning, conferred for example by Hox genes and Krox-20 expression (Wilkinson, et al., 1989; Krumlauf, 1994), establishing odd and even numbered rhombomeres, together with dorsoventral patterning, conferred for example by the expression of Pax genes (Goulding et al., 1993; Simon et al., 1995; Mansouri et al., 1994 for review) could result in specific surface properties of the basal lamina of the neural tube, retaining crest cells at the site of the exit points. Even numbered rhombomeres, in which the dorsoventral orientation has been inverted, generate their exit point in the former basal plate, now in alar plate position, at the correct dorsoventral location (Simon et al., 1995). In this respect, it is interesting to note that the exit point neural crest population expresses the cell adhesion molecule c-cad7. Cell adhesion involving cadherins has mainly been described as homophilic and c-cad7 expression could simply lead to the cohesion or coalescing of exit point cells, since there is no c-cad7 expression at the corresponding sites in the neural tube. However, heterophilic binding amongst different cadherins and probably also binding of cadherins to other cells surface proteins has recently been shown to be biologically important (Redies and Takeichi, 1993; Williams et al., 1994). Possibly an as yet unknown member of the cadherin family is expressed in the neural tube at the exit point site that acts in retaining c-cad7 expressing neural crest cells there. Also the expression of c-cad7 in exit points and then later in the motor neurons of the motor column which have exit points as their Fig. 7. Quail-chick chimaeras, double stained using c-cad7 in situ hybridisation and immunohistochemistry with the QCPN antibody. (A) Transverse section through the facial exit point of a homotopically grafted quail-chick chimaera showing the neural crest cells at the site of c-cad7 expression. (B) Section through facial exit point of a heterotopically grafted quail-chick chimaera where spinal cord crest had been transplanted into r4. The ectopic spinal cord crest cells express the appropriate marker, c-cad7 for their new environment. Scale bar, 50 µm. intermediate target, might constitute a recognition system with a function in axon guidance to the intermediate target of the exit points. We have focused our study on the exit/entry points of mixed motor-sensory nerves in the hindbrain and have identified a population of crest cells defining this region. These cells are specific for the region in which axons cross from the CNS into the periphery and vice versa. However, they are not necessarily restricted only to the branchiomotor nerve exit points. c- cad7, for example, is associated with several cranial motor nerve exit points, like the oculomotor and abducens nerve. Our preliminary evidence suggest that the abducens exit points, which are rootlets on the ventral side of the neural tube in rhombomere r5 and r6, are also defined by late emigrating neural crest cells. c-cad7 is also associated with dorsal root ganglia and spinal motor nerves, as is Krox-20 (Wilkinson et al., 1989; Schneider- Maunoury et al., 1993). However, Krox-20 expression in the trunk is associated with Schwann cell development (Topilko et al., 1994). Whether similar mechanisms are involved in the generation of nerve exit/entry points in the head and the spinal cord remains to be investigated. We wish to thank Dr Cairine Logan and Dr Isobel Heyman for a picture of the Krox-20 in situ hybridisation and the use of unpublished data. 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