THE JOURNAL OF COMPARATIVE NEUROLOGY 311:300-312 (1991)

Neuronal Differentiation and Maturation in the Mouse Trigeminal Sensory System, In Vivo and In Vitro DIDIER Y.R. STAINIER AND WALTER GILBERT Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02 138

ABSTRACT We have isolated and characterized four monoclonal antibodies (mAbs B33, E1.9, B30, and B10) that recognize mouse trigeminal sensory neurons at specific times during development. These antibodies permit the study of neuronal differentiation, axon outgrowth, and neuronal maturation in the trigeminal sensory system. With B33, we can follow migrating neural crest and placode cells into the anlagen of the trigeminal ganglion. E1.9 immunoreactivity marks neuronal differentiation and appears in the central nervous system at embryonic day 8.5 (E8.5) and in the peripheral nervous system at E9. E1.9 and B30 show the axonal outgrowth of trigeminal sensory neurons and reveal the pioneering of the peripheral tracts by an early population of ganglionic neurons. At this stage, in the central nervous system, mesencephalic trigeminal neurons are also E1.9 and B30 positive as they migrate to their final location in the rostra1 metencephalon. B30 and B10 allow us t o follow the maturation of these neurons. Also, in about 1%of the embryos, we identified mispositioned or misrouted trigeminal neurons. Furthermore, these biochemical markers facilitate the study of neuronal development in vitro. We find that, based on morphological and biochemical criteria, the maturation of trigeminal neurons in culture is target independent. Key words: monoclonal antibodies, axon guidance,ectopic neurons, neuronal morphology

During the development of the vertebrate nervous system, one of the first neuronal elements to differentiate is the trigeminal system. It consists of a motor nucleus and two populations of primary sensory neurons, the trigeminal ganglion and the centrally located mesencephalic trigeminal nucleus (MesV).The trigeminal sensory neurons convey information to the central nervous system (CNS) from a variety of sensory receptors in the periphery, mainly mechanoreceptors, thermoreceptors, and nociceptors in the face and the oral and nasal mucosa. Its central processes terminate on several groups of neurons in the brainstem, namely the principal sensory nucleus and the nuclei of the spinal trigeminal tract, which in turn project to the somatosensory cortex via relays in the thalamus (Paxinos, '85). In development, the peripheral sensory neurons originate from the mesencephalic neural crest and from the epibranchial placode (reviewed in mammals by Verwoerd and van Oostrom, '79) and the MesV neurons from the mesencephalic neural crest (as shown in birds by Narayanan and Narayanan, '78). In the mouse, the trigeminal ganglion becomes discernible by embryonic day 9 (E9) and sends fibers to its peripheral target from E9.5 to E l 3 (reviewedby Davies, '88).

o 1991 WILEY-LISS, INC.

Because of its clear definition and easy accessibility, the trigeminal ganglion has been widely studied in mammals and birds, yet our understanding of the cellular and biochemical events associated with neuronal differentiation, maturation, and axon guidance in the trigeminal system is limited to a few observations. Lumsden and Davies ('83, '86) examined mouse trigeminal axonal outgrowth in explant coculture studies. They suggested that sensory axons are guided by target-derived chemotropic factors: explicitly, that the early trigeminal target field releases a diffusible factor that specifically directs the growth of early trigeminal neurites. Studies by Moody et al. ('89b) in the chick trigeminal mesenchyme show that laminin, a permissive substrate for axon outgrowth in culture (Sanes, '851, is restricted to trigeminal axon pathways, whereas other extracellular matrix molecules exhibit a more general distribution. More recently, we showed that an early population of ganglionic neurons pioneers the peripheral tracts and that the later fibers extend along these pioneers. (The term Accepted May 14,1991. Didier Y.R. Stainier's present address is Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02114.

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NEURONAL DIFFERENTIATION IN TRIGEMINAL SYSTEM "pioneer" is used merely to suggest that these are the first detected axons in these pathways and that secondary or trailing fibers grow along them. No claim is made at this point about the requirement of these axons for the guidance of subsequent fibers). By contrast, in the CNS, MesV neurons extend towards the rhombencephalon independently, ignoring preexisting fibers (Stainier and Gilbert, '90). We used four specific monoclonal antibodies (mAbs) to describe the differentiation and maturation of mouse trigeminal sensory neurons and the pathways taken by their axons. MAb B33 recognizes a specific ganglioside expressed in the embryonic nervous system. MAb E1.9 recognizes a cytoplasmic epitope expressed only in primary sensory and motor neurons duringaxonal outgrowth, from E8.5 to E12. MAb B30 (Stainier and Gilbert, '89; Stainier et al., '91) recognizes a rare ganglioside expressed on trigeminal sensory neurons shortly after differentiation. MAb B10 recognizes another cytoplasmic epitope expressed only in the E1.9 immunoreactive subset of neurons after E1.9 immunoreactivity disappears. These four biochemical markers outline mouse trigeminal sensory neurons and their processes at various developmental times. They allow us not only to study neuronal differentiation and maturation but also to follow axonal outgrowth in a most informative wholemount preparation. In this report, we describe the temporal and spatial development of the trigeminal sensory neurons. We also study the differentiation and maturation of trigeminal neurons in culture and report that their maturation is target independent.

MATERIALS AND METHODS Monoclonal antibody production MAbs E1.9, B33, B30, and B10 were isolated from different fusions in which the immunogen consisted of whole cells from brains of different embryonic age: E1.9 from E l l mouse CNS, B33 and B30 from E l 5 rat mid- and hindbrains, and B10 from E l 8 mouse mid- and hindbrains. MAb production and screening was done as described previously (Stainier and Gilbert, '89). All four mAbs used in this study are mouse IgMs. MAbs E1.9, B30, and B10 were deposited with the Developmental Studies Hybridoma Bank and are available on request.

Immunohistochemistry Pregnant CD1 mice were obtained from Charles River under a specific breeding schedule: mice were bred for 3 h r from 12 noon to 3 p.m. on EO; our embryonic day ran from noon to noon. Animals were sacrificed by cervical dislocation or euthanized with halothane. Embryos, (E8-E12), staged according to Theiler ('891, were slit longitudinally along the forming neuropore and processed as half-embryo wholemounts. Brains of later embryos and postnatal animals were cut into 100-to 120-pm-thickvibratomesections and stained as previously described (Stainier and Gilbert, '89). All the staining reactions were done at room temperature (with gentle shaking) in polystyrene culture dishes by sequential incubations in the following: 10% normal goat serum (NGS) in phosphate-buffered saline (PBS) for one hr; hybridoma culture supernatant overnight (12-18 hr); after three 10-minwashes with 10% NGS/PBS, fluorescein-

ated goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL), diluted 1:100, for 2 hr. After three more 10-min washes in 10% NGS/PBS, the wholemounts and thick sections were mounted between two coverslips separated by silicone grease, in fresh mounting medium. For E1.9 and B10 staining, the tissue was fixed with 1% paraformaldehyde for 10 min, rinsed several times with PBS, and incubated for 2 h r in 10% NGS. Saponin, 0.04%, was included throughout the E1.9 and B10 immunostaining. We used a Lasersharp MRC-500 confocal microscope mounted with fluorescence optics to observe specific axonal pathways. Briefly, serial sections were collected and combined by projection. The resulting images were stored in a WORM optical disk (Maxtor Corporation, San Jose, CA) and printed using a video printer.

Cell culture and staining E9.5-E 10 mouse trigeminal ganglia were dissected and triturated, and the cells were plated on a polyornithine/ laminin substratum in plastic tissue culture dishes and grown in F12 medium supplemented with 10%horse serum and 5% calf bovine serum. Live cultures were stained as described (Stainier and Gilbert, '89) with B30 and B33. For E1.9 and B10 staining, the cultures were fixed with paraformaldehyde and permeabilized with the inclusion of 0.04% saponin throughout the procedure.

RESULTS Specific biochemical markers In vivo and in vitro, all the trigeminal sensory neurons stain with mAbs B33, E1.9, B30, and B10, in this order of appearance as the neurons differentiate and mature. MAb B33 recognizes the GD3 ganglioside as shown by thin-layer chromatography (TLC) blot analysis of acidic glycolipids (Stainier, '90). It labels neural crest and placode cells as well as crest- and placode-derived neurons. GD3 is also found in the developing CNS on the surface of both immature neuroectodermal cells and some mature neurons (Goldman et al., '84; Reynolds and Wilkin, '88). Figure 1A shows the B33 staining pattern in the trigeminal ganglion of an E l 0 mouse. At this stage, GD3-expressing cells include undifferentiated cells from the neural crest and placode as well as occasional neurons. MAb E1.9 recognizes a cytoplasmic epitope in a restricted population of neurons. This antibody is a marker for neuronal differentiation, since the filamentous E 1.9 immunoreactivity (IR) is expressed at the time of initial axon outgrowth and disappears when the axon stops growing. In the mouse, E1.9 IR appears at E8.5 and disappears by E12. E1.9 IR neurons comprise motor neurons of the ventral root, sensory neurons of the dorsal root ganglia (DRG), and neurons of the peripheral nervous system (PNS) including sensory and motor neurons of the cranial nerves. Figure 1B shows some E l l mouse trigeminal neurons stained with E1.9. The high resolution of confocal microscopy reproduces the filamentous nature of E1.9 IR and suggests the association of the E1.9 epitope with the cytoskeleton of the cell soma and its projections. MAb B30 recognizes a rare ganglioside on the surface of neural-crest- and placode-derived primary sensory neurons in the mouse (Stainier and Gilbert, '89; Stainier et al., '91). These include the sensory neurons of the trigeminal system

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Fig. 1. Trigeminal sensory neurons at different stages of their development are recognized by different monoclonal antibodies. A E l 0 mouse embryo stained with mAb B33 and viewed at the level of the trigeminal ganglion. At this stage, B33 IR cells include undifferentiated (neural crest and some placode) cells as well as occasional neurons and their processes. Scale bar = 50 pm. B: E l l mouse embryo stained with mAb E1.9 and viewed at the level of the trigeminal ganglion. At this stage, trigeminal neurons are bipolar and E1.9 IR. The fine optical dissection of the stained tissue allowed by the confocal microscope reveals the association of the EL9 epitope with the cytoskeleton of the

cell soma and its projections. Scale bar = 10 pm. C: Parasagittal section of a P6 mouse brain stained with mAb B30. In this confocal microscope reconstruction, B30 IR MesV neurons can be seen projecting independently in a common direction. Scale bar = 50 pm. D: A single B10 IR MesV neuron (from an E l 5 mouse) is dissected with the confocal microscope with increments of 2.5 pm. A n unusual degree of structural detail appears and reveals the association of the B10 epitope with the cytoskeleton of the cell soma as well as with the axonal projection. Scale bar = 10 pm.

both in the periphery and in the CNS. B30 outlines these neurons and their projections shortly after initial axonal outgrowth and until about 2 weeks postnatally. [The B30 ganglioside also appears in the cerebellum on the premigratory granule cells during the first postnatal week and on Purkinje cells by postnatal day 12 (P12) (Stainier and Gilbert, '89)l. The B30 ganglioside has recently been shown to play a role in the cellular adhesion process in vitro (Stainier et al., '91). Figure 1C shows some P6 mouse MesV neurons stained with B30. Clustered MesV neurons project independently in a common direction though occasionally two or more s o n s fasciculate for a short distance. MAb B10 recognizes another cytoplasmic epitope expressed specifically in the E1.9 IR subset of neurons. It is expressed, after E1.9 disappears, starting at E l 3 in the mouse. Its appearance within a population of neurons, such as a single DRG, is gradual, reflecting the gradual maturation of the neurons within a ganglion. Figure 1D analyzes a

single large MesV neuron in a thick section (100 Fm) of an E l 5 mouse CNS stained with mAb BlO, and dissected with a confocal microscope. An unusual degree of structural detail appears and clearly shows the association of the B10 epitope with the neuronal cytoskeleton. This B10 IR MesV neuron is also representative in its staining pattern of B10 IR neurons of the trigeminal ganglion.

In vivo neuronal differentiation and maturation Trigeminal ganglion. The trigeminal (or V) ganglion arises both from neural crest and from placodal epithelium (Narayanan and Narayanan, '80; D'Amico-Martel and Noden, '80). Neural crest precursors, B33 immunoreactive (Stainier, '901, migrate early (4- to 7-somite stage; E8) into the anlagen of the trigeminal ganglion, while placodal cell precursors, also B33 positive (Stainier, 'go),migrate slightly

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Fig. 2. Trigeminal ganglion of an E9 (20-somite-stage) mouse embryo stained with mAb E1.9. A few axons are emerging at the dorsal tip of the ganglion and form the budding ophthalmic projection ( 0 ) . These fibers are close together yet their growth cones are distinct, probing the environment in the general direction of growth. More ventrally (arrowheads), fibers span the ganglionic space; these will pioneer the maxillary and mandibular projections. D, dorsal; A, anterior. Scale bar = 50 Gm.

later (10-somite stage; E8.5) (Venvoerd and van Oostrom, '79). Placodal cell precursors are the first to differentiate and give rise to the earliest trigeminal neurons while neural crest precursors differentiate at a later stage to give rise to glia, satellite cells, and neurons of theV ganglion (Verwoerd and van Oostrom, '79; Chan and Tam, '88). The trigeminal and facial crest become distinct in the area of the cranial neural crest at E8.5 (8 to 12 somites) (Billingham and Silvers, '60; Chan and Tam, '88). At this stage, there is no detectable E1.9 IR in the trigeminal ganglionic region although E1.9 positive neurons are appearing in the mesencephalon (Stainier and Gilbert, '90). At the 14-somitestage, during E9, a few cells in the region of the primitive trigeminal ganglion are EL9 IR (Stainier and Gilbert, '90). This marks the onset of neuronal differentiation and is consistent with the [3H]thymidinebirthdating studies in the rat trigeminal ganglion (Altman and Bayer, '82) and the electron microscope observations in the mouse (Davies and Lumsden, '84). Bipolar neurons send short projections towards the periphery and the neural tube. By the 20-somitestage (E9-E9.5)' the ophthalmic projection (0)is condensed and directed (Fig. 2); the growth cones stand close together, yet these early axons grow independently of each other (see also Stainier and Gilbert, '90). Shortly after neuronal differentiation, starting at E9.5, B30 IR outlines trigeminal neurons. Figure 3 shows sparsely distributed bipolar neurons spanning the region of the primitive V ganglion. These large, fusiform-shaped trigeminal neurons extend processes both into the neural tube at the level of the pons and out to their peripheral targets.

Fig. 3. Trigeminal ganglion region of an E9.5 (27-somite-stage) mouse embryo stained with mAb B30. Sparsely distributed B30 IR neurons exhibit a bipolar morphology with fine neurites growing in opposite directions from elongated spindle-shaped cell bodies. They span the region of the primitive trigeminal ganglion. Ophthalmic

neurons at the dorsal side of the ganglion (arrowheads) are projecting dorsally towards the mesencephalon while the maxillomandibular neurons project in the anteroposterior axis. Inset: High magnification view of several B30 IR bipolar neurons. D, dorsal; A, anterior. Scale bars = 50 pm; inset = 10 pm.

304 Starting at El0 and spanning a period of about 48 hr, a second wave of neurogenesis populates the V ganglion as revealed by a sharp increase of E1.9 and B30 IR neurons, and in agreement with the [3H]thymidinebirthdating studies in the rat trigeminal ganglion (Forbes and Welt, '81; Altman and Bayer, '82). As more neurons differentiate from neural crest precursors within the ganglionic space, fasciculating peripheral axons exit the ganglion as thickening bundles. The maxillary and mandibular projections, initially distributed throughout most of the ganglion, now segregate themselves; the ophthalmic projection, a comparatively minor one, remains distinct at the dorsal tip of the ganglion throughout development. Axons that have not joined a major bundle inside the ganglionic space quickly do so once they exit the ganglion. This segregation is reflected inside the ganglion where most neurons are directly adherent to a major bundle. Figure 4 illustrates the organization of an E11.5 trigeminal ganglion as revealed by the B30 immunostaining of a wholemount preparation. Inside the ganglion, individual neurons stay clustered, closely adherent to thickening projections. These projectionsthen gather into major bundles that project out of the ganglion. At the end of axon outgrowth by E12, E1.9 IR disappears, but B10 IR appears and B30 IR persists. By now, the V ganglion has become a cohesive structure with three very

Fig. 4. E11.5 mouse embryo stained with mAb B30 and viewed at the ventral region of the trigeminal ganglion. A: Thickening bundles of axons span the ganglion and gather at its periphery to form the trigeminal projections. B: Inside the ganglion, individual neurons stay clustered and their axons quickly join an adjacent thickening bundle. Neighboring neurons seem to join the same bundle. Scale bars = 50 p,m.

D.Y.R. STAINIER AND W. GILBERT

Fig. 5. Whisker pad area of an E14.5 mouse: parasagittal slice of the head stained with mAb B30. B30 IR branches of the maxillary projection surround individual vibrissae follicles at a time of innervation. Scale bar = 50 am.

distinct and compact nerves, each projecting to a defined area. For example, in Figure 5, the maxillary nerve terminates in the whisker pads area of an E14.5 mouse. At this time of innervation, B30 IR maxillary branches have surrounded individual whisker follicles. Mesencephalic trigeminal nucleus. Neurons of the MesV are thought t o derive from the mesencephalic neural crest (Narayanan and Narayanan, '78; Stainier et al., '91). They constitute the only primary sensory neurons in the CNS and their differentiation parallels that of the V ganglion neurons. Mesencephalic neural crest cells emerge early; they are born and start migrating between the 4- to 7-somite stage embryos (E8) (Nichols, '86; Chan and Tam, '88).At E8.5, by the 10-somite stage, EL9 IR first outlines a few MesV neurons in the rostral part of the mesencephalon (Stainier and Gilbert, '90). MAb B30, which we showed to be specific for MesV neurons in the mouse CNS (Stainier and Gilbert, '89), first outlines such caudally projecting neurons at a slightly later stage in their maturation. Figure 6A shows an E9.5, 24-somite-stage embryo, stained with B30. B30 immunostaining reveals that MesV neurons have, by this stage of differentiation, projected caudally to the second rhombomere where they exit the CNS. Their axons will then mix with the neurons and axons of theV ganglion and follow the trigeminal fibers, mostly of the mandibular branch, to their respective peripheral targets. At this stage, most MesV neurons appear bipolar and they span a vast area of the mesencephalon. A low power sketch of the right mid- and hindbrain region of an E l 0 mouse embryo illustrates the location of the migrating mesV neurons and their projections (Fig. 6B). The same region is shown stained with mAb E1.9 in Figure 7. MesV neurons are E1.9 IR as well as oculomotorand trochleomotorneurons that stand rostromedially. At the 40-somite stage (Ell), EL9 IR starts to disappear but B30 IR will persist until 2 weeks after birth. The B30 staining allows us to witness the extensive migration of the MesV neurons from the mesencephalic neural folds to their final location in the rostral metencephalon. The migration of these cells in the direction of their axonal projection occurs in parallel with their maturation into pseudounipolar neurons and is mostly completed by E13. In a specific staining pattern that parallels that of B30, mAb B10 labels, in the CNS, MesV neurons as well as

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mes

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Fig. 6. A: E9.5 (24-somite-stage)mouse embryo stained with mAb B30 and viewed at the level of the mid- and hindbrain. B30 IR MesV neurons differentiate in the mesencephalon and send axons caudally to the second rhombomere where they exit the CNS. Most MesV neurons are bipolar, showing a trailing process. Inset: High magnification view of the area marked by the arrow. Individual MesV neurons can be identified by their B30 IR. They quickly differentiate upon leaving the mesencephalic neural folds (top) and migrate in the direction of their axonal projection to settle in the rostral metencephalon. B: Schematic drawing illustrating the migration of the MesV neurons from the mesencephalic neural folds (mes) to the rostral metencephalon (met) (asterisk) in the direction of their aional projection. D, dorsal; A, anterior; 111, third cranial (or oculomotor) nerve; IV,fourth ventricle; dienc, diencephalon; TG, trigeminal ganglion. Scale bars = 50 pm; inset = 10 pm.

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Fig. 7. E l 0 (30-somite-stage) mouse embryo stained with mAb E1.9 and viewed at the level of the head. In the CNS, E1.9 IR neurons include mesencephalic trigeminal nucleus (MesV) neurons as well as motor neurons of the third (oculo), fourth (trochlear), and fifth (trigeminal) cranial nerves. Again, E 1.9fibers extend from the mesencephalic neural folds to the second rhombomere outlining the projection and migration

of MesV neurons. Outside the CNS, branches of the third nerve (111) and neurons of the trigeminal ganglion (TG)are also E1.9 IR. Posterior to the ophthalmic projection, an extra projection (arrow) branches out after a short ectopic outgrowth. D, dorsal; A, anterior; IV, fourth ventricle. Scale bar = 50 pm.

oculomotor and trochleomotor neurons. The BlO cytoplasmic epitope is present starting at E l 3 well into postnatal life and outlines the cytoskeleton of specific neurons including their axonal and dendritic processes. Figure 8A is a horizontal section of an E l 5 mouse brain stained with B10. We can identify two main populations of B10 IR neurons in this plane: motor neurons of the third cranial nerve (open arrows), also shown in the inset, and MesV neurons (filled arrows), also shown in Figure 1D. Figure 8B shows a parasagittal section of a P6 mouse brain stained with B10. MesV neurons as well as their axons and collaterals are stained and the B10 1R axons can be followed for several millimeters. Description of the postnatal organization and maturation of the MesV nucleus was the focus of an earlier study (Stainier and Gilbert, '89).

the ophthalmic projection. After a short (50 pm) outgrowth, it stops and starts branching. Here, branching occurs in vivo in the absence of recognizable target tissue. We observed this pattern once in approximately 80 or more embryos. In another case, a B10 IR MesV neuron stands medially in the cerebellar space of a P3 mouse; its axon starts growing rostrally towards the aqueduct for a short distance, and then heads ventrocaudally (Fig. 9). We noted such a misplaced MesV neuron only once in 100 or more early postnatal brains. Late prenatally, a period of extensive cell death reduces by half the number of neurons in the MesV nucleus (Alley, '74). Although we could not directly analyze the connectivity of that B10 IR MesV neuron, its mere survival in such a grossly ectopic position suggests that its axon has reached a supporting target.

Ectopic neuronal location and/or outgrowth During the course of our study, we came across a few cases of ectopic neuronal location and outgrowth in the trigeminal system. Such cases, revealed by our mAbs, are informative by themselves because they indicate what the system can accomodate. It is also important to record phenotypic variance in a wild-type background in order to characterize properly the phenotype of mouse mutants. One case of ectopic outgrowth is shown in Figure 7; an extra projection (arrow) spurts out of the V ganglion posterior to

In vitro differentiation and maturation of trigeminal neurons Trigeminal sensory neurons go through an identifiable sequence of morphological and biochemical maturational events as revealed by our mAbs. In vivo, B33 IR neural crest and placode precursors give rise to trigeminal sensory neurons. Newly differentiated E1.9 IR trigeminal neurons (Fig. 1B) are truly bipolar and their processes extend for 1 to 2 days. Shortly after differentiating, they become B30 IR.

Fig. 8. A Horizontal section of an E l 5 mouse embryo stained with mAb BlO. B10 IR MesV neurons (filled arrows) surround the lateral borders of the aqueduct (Aq). Motor neurons of the third cranial nerve are also B10 IR (open arrows). Scale bar = 300 km. Inset: High magnification view of two B10 IR motor neurons of the third cranial nerve. Confocal microscope projection of several focal planes. Scale bar = 10 km. B: Parasagittal section of a P6 mouse brain stained with

mAb B10. MesV neurons and their axons are clearly B10 IR. The mesencephalic trigeminal nucleus can be divided into dorsal (asterisk) and ventral (arrow) parts. In the caudalmost section of the ventral part, cells extend into the peduncles of the cerebellum (Cb). See Stainier and Gilbert, '89 (Fig. 2) for a similar parasagittal section stained with mAb B30. D, dorsal; P, posterior. Scale bar = 300 pm.

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Fig. 9. A Horizontal section of a P3 mouse stained with mAb B10. B10 IR MesV neurons (arrowheads) surround the lateral borders of the aqueduct (Aq). Medially, in the cerebellar space (Cb),a single B10 IR

neuron stands in an ectopic position (arrow). Scale bar = 300 pm. B. This ectopic B10 IR neuron first extends rostrally, and then ( C ) turns around and projects ventrocaudally. Scale bars = 50 pm.

As they stop growing, the processes gradually join together close to their points of origin as a result of asymmetrical growth. These processes also send out collaterals and start branching more heavily at their distal end. The maturing trigeminal neurons, with their characteristic T-shaped process, become B10 IR about 4 days after differentiation. In vitro differentiation and maturation of trigeminal sensory neurons reflect the in vivo sequence. Newly differentiated neurons are E1.9 IR. Indeed, one frequently observes pairs of E1.9 IR neurons that have started extending axons before the completion of cytokinesis (data not included). MAb B30 stains them shortly thereafter. Figure 1OA shows a B30 IR trigeminal sensory neuron 12 hr after plating. In these young cultures originally seeded with E9.5-El0 trigeminal cells, neurons exhibit a bipolar morphology with fine unbranched neurites growing in opposite directions from small spindle-shaped bodies. Twenty-four hours after plating, these neurons start branching and send simple collaterals along their axons, as shown in Figure 10B. B10 IR appears about 5-7 days after plating and stains the neuronal population gradually, as observed in vivo. More complex branching patterns of the processes also appear by the end of the first week in culture (Fig. 10C,D) and some neurons even become pseudounipolar (Fig. 10E). These trigeminal sensory neurons show extensive branching in the absence of target tissue; furthermore, the

addition of NGF (50 ng/ml) to such cultures does not alter the timing or the characteristics of the maturation process of these neurons. Regenerating (E13 and older) trigeminal neurons in culture grow out expressing the E1.9 antigen and only 5-7 days later regain B10 IR.

DISCUSSION Trigeminal sensory system The mouse trigeminal sensory system is an ideal system to study neuronal development. The clear definition and easy accessibilityof its peripheral component has facilitated the investigation of several aspects of neuronal differentiation (reviewed by Davies, '88). In vitro studies by Davies and Lumsden ('83, '86) have suggested the chemotropic guidance of the earliest sensory axons. Also, studies of the site and timing of nerve growth factor (NGF) synthesis and NGF receptor (NGFR) expression indicate that the commencement of NGF synthesis and NGFR expression coincides with the onset oftarget field innervation (Davieset al., '87). [A more recent study of NGFR mRNA expression shows that the NGFR gene is transcribed at a constant low level in the ganglion and its maxillary target field before the arrival of the earliest axons (Wyatt et al., ,9011. The development of a number of monoclonal antibodies specific

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Fig. 10. Differentiation and maturation of trigeminal neurons in culture. E9.5-El0 trigeminal ganglia were dissected, dissociated, and plated on a poly-ornithinenaminin substratum. The cultures were then stained with mAb B30 at various times after plating. Scale bars = 100 pm. A: Trigeminal neuron after 12 hr in culture. In these young cultures, neurons exhibit a bipolar morphology with fine unbranched neurites growing in opposite directions from small spindle-shaped bodies. At this point they are also EL9 IR. B. Trigeminal neuron after 24 hr in culture. Such neurons show simple branching at the distal end

of their projections and start sending collaterals along their axons or even from their cell body. They are still E1.9 IR. C:Trigeminal neurons after 5 days in culture. The branching at the distal end of their projections has become more complex. These neurons have lost the EL9 epitope and are now B10 IR. D and E. Trigeminal neurons after 7 days in culture. Numerous collaterals have grown at various points along the initial projections and branching occurs mainly at their distal ends. Some trigeminal neurons even become pseudounipolar such as the one shown in (E). Such neurons are BlO IR.

for mouse trigeminal sensory neurons has helped us characterize further the differentiation and maturation of this system. A detailed analysis of the earliest axonal outgrowth showed a pioneering phenomenon in the periphery whereas in the CNS MesV axons grow independently of each other (Stainier and Gilbert. '90). By delineating the' developmental events in the trigeminal ganglion and by providing biochemical markers for these events, this study sets the stage for the molecular characterization of the development of this neuronal sys-

tem. Furthermore, the description and availability of these biochemicalmarkers will help characterize the developmental phenotype of mouse mutants since the development of their primary sensory and motor systems can now be looked at in detail using mAbs E1.9 and B10.

Neuronal differentiation and maturation The differentiation of neurons in the trigeminal ganglion can be temporally divided into two broad periods. By day

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310 E9, a small number of neurons appear in the ganglionic space. This early population of neurons will pioneer the peripheral pathways. For about 24 hr, there will be little additional neuronal differentiation. Then, starting at E 10 and for about 48 hr, a second wave of neurogenesis takes place. The vast majority of the peripheral trigeminal neurons are born in this period; their number increases to reach a maximum of about 40,000 by E l 3 but reduces to about 20,000 by birth (Daviesand Lumsden, '84). Both the neural crest and the placode contribute to generate neurons in the V ganglion (Verwoerdand van Oostrom, '79; D'amicoMartel and Noden, '80) and this dual origin has been related to the temporal separation of neuronal differentiation (Moody et al., '89a). Cove11and Noden ('89) have in fact provided some evidence supporting Hamburger's claim ('61) that, in the chick, epidermal placodes give rise to the neurons that pioneer the different trigeminal pathways. In the central component of the trigeminal sensory system, the MesV nucleus, neural-crest-derived neurons differentiate at a steady rate. Their appearance as revealed by E1.9 IR precedes that of the peripheral trigeminal neurons. In fact, axonal branches of MesV neurons seem to be the first projections to descend from the midbrain towards the spinal cord, joined at the pontine region by afferents from the V ganglion. As has been shown in the frog and the zebrafish, trigeminal neurons are among the earliest ones born; their axons thus form part of the early scaffolding of the CNS (Nordlander et al., '85; Mendelson, '86). MAb EL9 and mAb B10 outline the same general subset of neurons at different stages of their development. Mab E1.9 stains primary sensory and motor neurons during axonal outgrowth, from E8.5 to E12; mAb B10 stains primary sensory and motor neurons starting at E13, when the axons have stopped growing and connections are starting to be formed. The distribution of the E1.9 and B10 antigen(s) is very similar to the distribution of a recently characterized type I11 intermediate filament (IF), peripherin or 57-kDa neural IF protein (Greene, '89; Escurat et al., '90). Neither E1.9 nor B10 gives consistent results on Western blots but a detailed double-labelling study of peripherin versus E1.9 and B10 points to their differences. E1.9 IR precedes peripherin expression and is not seen in either the optic or the olfactory system where peripherin is expressed (Stainier, '901, suggestingthat EL9 is not against peripherin. B10 IR appears after the onset of peripherin expression; it outlines a subset of peripherin-positive DRG neurons at various ages and is not seen in either the optic or the olfactory system (Stainier, '90). This distribution leaves unresolved the possibility that B10 might recognize a modified epitope of peripherin, although none of the peripherin antisera tested blocked B10 staining. The specific expression of the EL9 and B10 antigen(s) in a subset of neurons may reflect the evolutionary age of these old primary neurons versus more recently evolved forebrain structures. In fact genetic evidence in the zebrafish provides a molecular argument for such a distinction (Grunwald et al., '88). An alternative explanation, suggested by Escurat et al. ('88, '90) for the specific expression of peripherin, is that most of these neurons send axons outside the encephalic spinal cord.

Axon outgrowth At its earliest stage of neuronal differentiation (E9), the trigeminal ganglion already looks polarized: short fibers span its width and grow out towards the periphery (Stainier

and Gilbert, '90). The first organized outgrowth out of the ganglion is the ophthalmic projection (Fig. 2). Three to five leading axons grow dorsally towards the mesencephalon and then turn rostrally to extend around the eye cup. The mechanisms underlying this directed outgrowth have been addressed by two different though not exclusive models. Lumsden and Davies ('83, '86) have proposed a neurotropic model whereby the trigeminal cutaneous target field releases a diffusible factor that specifically directs neurite outgrowth from the trigeminal ganglion. Others have proposed a substrate guidance model after demonstrating a restricted distribution of laminin in the peripheral pathways of trigeminal axons (Riggottand Moody, '87; Moody et al., '89b). However, the demonstration that an early population of neurons pioneers the peripheral tracts during E9 (Stainier and Gilbert, '90) calls for a more detailed temporal analysis of these models. MAb B33 allowed us to look at the migration of neural crest cells into the anlagen of the trigeminal ganglion but also in the trigeminal and facial mesenchymes. This analysis prompts the discussion of a third axon guidance model according to which migrating neural crest cells would lead the earliest trigeminal sensory axons. This was originally suggested following Le Douarin's observation that the pathways of the migrating crest cells in the chick closely outline the pathways of the trigeminal sensory axons ('82). In the mouse, neural crest cells start migrating out of the columnar epithelium near the tips of the midbrain-rostra1 hindbrain neural folds at the 4- to 7-somite stage. Initially confined to the subectodermal region of the cranial mesenchyme, they then colonize the lateral craniofacial mesenchyme, the developingtrigeminal ganglion, and the pharyngeal arch (Nicholls, '81, '86; Chan and Tam, '88). These early migrating neural crest cells may lead the way for sensory pioneer axons. In fact, presumptive Schwann cells (neural crest derived) have been shown to migrate ahead of growth cones in chick limb buds (Keynes, '87; Noakes and Bennett, '87). Furthermore, early neural crest removal results in a defective sensory innervation of chick limbs (Carpenter and Hollyday, '86). Schwann cells and their precursors synthesize laminin (Cornbrooks et al., '83; Moody et al., '89b) and it is conceivable that these cells lay down a laminin trail. In fact, Moody et al. ('89b) indicate that in the chick embryo, trigeminal sensory axons seem to follow a route predefined by HNK-1 IR presumptive Schwann cells. This is specific for sensory fibers as latergrowing trigeminal motor axons do not exhibit this association. We have also noticed that, in vivo, migrating neural crest cells, B33 IR, seem to precede the earliest sensory fibers, and that, in explanted neural tube cultures, peripheral sensory fibers preferentially grow on B33-positive cells migrating away from these explants (data not included). The availability of a few mouse cephalic neural crest mutants such as Patch (Ph) and SpZotch (Sp) may allow one to assess further the role of migrating neural crest cells in the guidance of trigeminal sensory fibers.

Establishment of somatotopy The importance of fasciculation in establishing the topographic relationship between the periphery and the CNS during the development of the trigeminal system was suggested by Erzurumlu and Killackey ('83). The clear and gradual bundling of trigeminal axons seen in Figure 4 illustrates the concept of progressive fasciculation.Thickening fascicles originate within the ganglion and gather into

NEURONAL DIFFERENTIATION IN TRIGEMINAL SYSTEM major bundles that project out to their peripheral targets. Inside the ganglion, it appears that neighboring neurons join a common fascicle. If the relative order and cohesion of large fascicles is maintained [as supported by Erzurumlu and Killackey ('83), but also see Davies and Lumsden, '861, progressive fasciculation provides a simple model for the somatotopy observed in the embryonic trigeminal system (Van der Loos and Welker, '85; Rhoades et al., '90): discrete groups of neurons in the ganglion would project to specific target areas both in the PNS and in the CNS thus creating a primary or crude map. The specificity of projection would arise by selective fasciculation on the earliest or pioneering fibers (Stainier and Gilbert, '90). The final map would then be formed by the selective maintenance of topographically related projections in the peripheral and central target fields of the trigeminal ganglion as proposed by Davies and Lumsden ('86). Because of the conflicting results on the maintenance of order in the trigeminal maxillary nerve (Erzurumlu and Killackey, '83; Davies and Lumsden, '861, the labelling of closely neighboring ganglion cells by a tracer, such as the fluorescent carbocyanine dye di-I, should reveal the path of individual axons throughout the nerve. This would then indicate whether pattern transfer is due to the maintenance of order in the developingtrigeminal nerve or to the sorting out of the individual projections as they approach the target, as may be the case in the visual system (Colelloand Guillery, '90). Also, as the B30 ganglioside plays a role in the cellular adhesion process (Stainier et al., '91), it may be involved not only in holding together peripheral sensory structures, such as the trigeminal ganglion, but also in the pronounced fasciculation of trigeminal fibers.

In vitro maturation of trigeminal sensory neurons Using a number of morphological and biochemical criteria, we showed that the maturation of sensory neurons from the trigeminal ganglion is target independent. When Chamak et al. ('87) looked at CNS neurons in culture, their results suggested that the astrocytic environment regulates microtubule-associatedprotein 2 (MAP2) IR as well as the morphology of the neurons, and that this regulation is region specific. Likewise, cells intrinsic to the trigeminal ganglion are sufficient to regulate the branching and the appearance of the B10 antigen in cultured trigeminal neurons. This stands in contrast to the plasticity of the neurotransmitter phenotype of sympathetic neurons and to the role of environmental factors (including the targets) in determining transmitter choice (reviewed by Landis, '90). NGF, one of the neurotrophic factors for peripheral trigeminal neurons (Ebendal and Hedlund, '75), did not speed up the appearance of B10 IR in trigeminal neurons dissected early nor did it induce B10 IR in PC12 cells (data not included). This is not surprising as the survival and growth of trigeminal neurons is independent of NGF when their s o n s are growing towards their targets, and these neurons only become responsive to NGF after their arrival into, and their exposure to the target field (Lumsden and Davies, '83; Davies and Lumsden, '84; Wyatt et al., '90). Also, by branching extensively in both their peripheral and central target areas, the trigeminal neurons establish a highly interwoven and redundant network that can be refined to somatotopy by the selective maintenance of topographically related connections.

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ACKNOWLEDGMENTS We thank M. Goldman, L. Parisek, and J. Gorham for providing peripherin antiserum and C. Fulwiler and M. Grether for critical reading of the manuscript.

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