Role of the Target in the Pathfinding of Facial Visceral Motor Axons

MCN

Molecular and Cellular Neuroscience 16, 14 –26 (2000) doi:10.1006/mcne.2000.0855, available online at http://www.idealibrary.com on

Role of the Target in the Pathfinding of Facial Visceral Motor Axons John Jacob,* ,1 Marie-Catherine Tiveron, †,1 Jean-Franc¸ois Brunet, † and Sarah Guthrie* ,2 *MRC Centre for Developmental Neurobiology, King’s College, Guy’s Campus, 4th Floor New Hunt’s House, London SE1 9RT, United Kingdom; and †Laboratoire de Ge´ne´tique et Physiologie du De´veloppement, Institut de Biologie du De´veloppement de Marseille, CNRS/INSERM/Universite´ de la Me´diterrane´e, IBDM Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France

Axon navigation depends, in part, on guidance cues emanating from the target. We have investigated the possible role of the target in the pathfinding of visceral motor axons to cranial parasympathetic ganglia. Mice homozygous for a tau-LacZ transgene targeted in the Phox2a locus lack the sphenopalatine ganglion, which is the normal target of visceral motor axons of the facial nerve. We found that in these mutants, facial visceral motor axon pathfinding was disrupted, and some axons were misrouted to an alternative parasympathetic ganglion. Moreover, the absence of correct facial visceral motor pathways was concomitant with defects in the pathfinding of rostrally-projecting sympathetic axons.

INTRODUCTION Precise axon pathfinding depends on guidance molecules, which act in a contact-mediated or diffusible manner and may be growth-inhibiting, repulsive, growth-promoting, or chemoattractant (TessierLavigne and Goodman, 1996). Sources of chemoattractant cues may include intermediate or final axon targets. Several target-derived diffusible guidance cues have recently been characterized, including neurotrophins produced in the branchial arch which chemoattract trigeminal sensory axons (O’Connor and TessierLavigne, 1999). We have tested the hypothesis that target ganglia provide guidance cues for preganglionic parasympathetic motor neurons of the brain stem. These neurons 1

These authors contributed equally to this work. To whom correspondence should be addressed. [email protected]. 2

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provide the major outflow of the parasympathetic nervous system and will be referred to as visceral motor (VM) neurons. VM neurons extend axons via the oculomotor, facial and glossopharyngeal cranial nerves, to innervate peripheral neural crest-derived autonomic ganglia in the head. In addition to VM neurons, the brain stem contains branchiomotor (BM) and somatic motor neuron classes. BM and VM axons navigate toward large dorsal exit points (Lumsden and Keynes, 1989), but later diverge to innervate branchial muscle and ganglionic targets, respectively (Lumsden, 1990; Simon et al., 1994; Studer et al., 1996). One or more of these cranial motor neuron classes contribute to discrete nuclei which occupy distinct positions along the rostrocaudal axis of the brain stem; in the hindbrain these nuclei develop within single rhombomeres or pairs of rhombomeres (Lumsden and Keynes, 1989). Thus the facial nerve contains BM and VM axons derived from r4 and r5 while BM and VM neurons that contribute to the glossopharyngeal nerve develop within r6 and r7. In the rodent embryo, facial BM neurons originate in r4, but migrate caudally to form the facial motor nucleus in r6 (Auclair et al., 1996; Studer et al., 1996; Pattyn et al., 1997). VM neurons occupy a discrete territory in ventrolateral r5, forming the superior salivatory nucleus (Auclair et al., 1996). Facial VM axons exit the hindbrain to project rostrally via the greater superficial petrosal nerve (Gspn) to their synaptic target, the sphenopalatine ganglion (Spg; Fig. 1A). Previous studies have shown that in the trunk, sympathetic preganglionic and postganglionic axons are able to pathfind successfully in the absence of their target, suggesting either that pathfinding in this system is cell autonomous or that guidance cues for sympa1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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thetic axons are derived from sources other than the target ganglia (Yip, 1987, 1990, 1996; Guillemot et al., 1993; Guidry and Landis, 1995). In contrast, little is known about the source of putative parasympathetic VM guidance cues. However, these two subdivisions of the autonomic nervous system utilize common transcriptional determinants along the pathway to differentiation (Guillemot et al., 1993; Bober et al., 1994; Anderson et al., 1997; Morin et al., 1997; Pattyn et al., 1999) and have similar trophic requirements (Buj-Bello et al., 1995; Kotzbauer et al., 1996; Baloh et al., 1998; Heuckeroth et al., 1999), perhaps implying that they also share a common target-independent axon guidance mechanism. In order to determine whether the target plays a role in parasympathetic VM axon pathfinding, we analyzed the facial VM pathway in mouse embryos with a targeted null mutation of the paired-like homeodomain transcription factor, Phox2a (Morin et al., 1997). These mutants lack the sphenopalatine ganglion which is the synaptic target of facial VM axons. Peripherally, Phox2a is expressed by neurons in all divisions of the autonomic nervous system, while in the central nervous system it is expressed in all brain-stem noradrenergic centers and in several motor nuclei, including the facial nucleus (Tiveron et al., 1996). Previous studies showed that in embryos with a null mutation of Phox2a all cranial parasympathetic ganglia, including the sphenopalatine ganglion, are absent or atrophic (Morin et al., 1997). The mutant phenotype correlates with only a subset of the sites of Phox2a expression, probably due to coexpression of a closely related transcription factor, Phox2b, which is able to compensate for the absence of Phox2a in some cell types but not in others (Pattyn et al., 1997; 1999). We examined transgenic mice harboring a tau-lacZ marker gene in the Phox2a locus, enabling visualization of peripheral axonal pathways of all neurons expressing Phox2a. We found that in the absence of the sphenopalatine ganglion, VM axons from r5 failed to pathfind correctly. Specifically, the Gspn was absent or was evident only as a stump. Moreover, VM axons were later rerouted, forming an ectopic projection to a different cranial parasympathetic ganglion, suggesting that growth cones select the most appropriate target from a hierarchy of alternative preferences. In addition to Phox2a, the sphenopalatine ganglion is dependent on the related homeobox gene Phox2b (Pattyn et al., 1999) and the bHLH transcription factor Mash1 (Guillemot et al., 1993). Phox2b ⫺/⫺ mice lack both parasympathetic ganglionic and VM neurons (Pattyn et al., 2000). In Mash1 ⫺/⫺ mice, which have no obvious defects of cranial motor neurons (Hirsch et al., 1998), a sphenopalatine ganglion initially forms and later de-

generates. The presence of this structure, albeit transient, allowed us to determine whether Mash1 has a role in regulating putative sphenopalatine ganglion-derived guidance cues, by examining Gspn development. We found that in these embryos, the Gspn was always present, indicating that VM axon pathfinding requires a sphenopalatine ganglion-derived factor (or factors), which is not regulated by Mash1.

RESULTS Autonomic Pathways in the Head Facial VM and BM axons leave the brain to form the intermediate nerve and the facial motor root, respectively, subsequently converging at the distal sensory ganglion of the facial nerve, the geniculate ganglion. At this point a large contingent of VM axons turns rostrally to form the Gspn, which extends along the maxillary division of the trigeminal nerve toward the sphenopalatine ganglion. The Gspn also contains axons of sensory neurons whose cell bodies lie within the geniculate ganglion. Growing caudally from the geniculate ganglion, the facial nerve (BM and VM) gives rise to the chorda tympani branch, in which remaining VM axons turn rostrally toward the submandibular ganglion in the first branchial arch. Thereafter, BM axons constitute the sole motor component of the facial nerve, which grows toward the second branchial arch (Fig. 1). A similar segregation of BM and VM pathways occurs in the glossopharyngeal nerve; only the VM pathway is described here. VM neurons from the inferior salivatory nucleus send their axons via a tympanic branch to the lesser deep petrosal nerve and the otic ganglion (Fig. 1). The parasympathetic pathway of the Gspn is anatomically related to sympathetic axon pathways, which arise from the superior cervical ganglion to project rostrally, forming a plexus around the internal carotid artery. These sympathetic axons associate with the Gspn at more rostral levels to form the vidian nerve. Only the parasympathetic axons synapse in the sphenopalatine ganglion, while the postganglionic sympathetic fibers course through en route to innervate the cranial vasculature (Fig. 1B). Facial VM Neurons Express Phox2a Mice with a null mutation of the Phox2a gene lack several parasympathetic ganglia, including the sphenopalatine ganglion. Since the sphenopalatine ganglion is innervated by a specific population of facial VM neurons, we used Phox2a mutant mice as a model to explore

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FIG. 1. Schematic diagram of cranial nerve nuclei and related autonomic projections in the mouse at E13.5 and E16.5. (A) E13.5. At this stage the vidian nerve has not yet formed. Superior cervical ganglion axons have reached the point of fasciculation with the greater superficial petrosal nerve. (B) E16.5. By this stage the vidian nerve consisting of sympathetic and parasympathetic axons has formed. Sympathetic axons course through the sphenopalatine ganglion, but do not synapse on postganglionic neurons. See text for details. Parasympathetic axons are shown in red, sympathetic axons in green, and BM axons in blue. For simplicity, only the maxillary division of the trigeminal nerve is represented as it is closely related anatomically to the sphenopalatine ganglion. The ophthalmic and mandibular branches are shown as dotted lines extending from the trigeminal ganglion. Abbreviations: TBM, trigeminal nucleus; FVM, superior salivatory nucleus; FBM, facial motor nucleus; GVM, visceral motor (inferior salivatory) nucleus of the glossopharyngeal nerve; In, intermediate nerve; Mtr, motor root of the facial nerve; Gln, glossopharyngeal nerve root; Tg, trigeminal ganglion; Gg, geniculate ganglion; Pg, petrosal ganglion; Maxn, maxillary nerve; Gspn, greater superficial petrosal nerve; Vn, vidian nerve; Ctn, chorda tympani; Facn, facial nerve; Ldp, lesser deep petrosal nerve; Spg, sphenopalatine ganglion; Smg; submandibular ganglion; Otg, otic ganglion; Scg, superior cervical ganglion; Ba2, second branchial arch.

whether the axonal guidance and/or outgrowth of these neurons depends on their ganglionic target. Phox2 genes are expressed in all pre- and postganglionic neurons of the parasympathetic and enteric nervous systems examined so far (Tiveron et al., 1996; Pattyn et al., 1997). We therefore sought to visualize the VM projections to the sphenopalatine ganglion in a Phox2a mutant background, by the use of a mouse line harboring a tau-lacZ transgene in the Phox2a locus. To validate this approach we first established that facial VM neurons express Phox2a. Facial VM neurons are born in the ventral aspect of r5 and migrate dorsolaterally, to form the superior salivatory nucleus. Thus far, two lines of evidence suggest that this nucleus is a site of Phox2a expression. First, we previously established that, at E10.5, postmitotic precursors in the r5 motor column express Phox2a (Pattyn et al., 1997, and submitted for publication). Second, in postnatal mice Phox2a-expressing neurons are found scattered in the rostral medulla, overlapping the lateral field of the reticular formation, in a pattern reminiscent of the distribution of facial VM neurons (Tiveron et al., 1996). However, it is difficult unambiguously to identify facial VM neurons at birth, since they do not form a discrete nucleus (Contreras et al., 1980; Tiveron et al., 1996). We sought to demonstrate directly the identity of Phox2a-expressing r5 ventral precursors by using

Phox2a immunofluorescence in combination with retrograde labeling of facial VM fibers using fluorescent dextran tracers. In sections through the r5 region, double-labeled Phox2a ⫹/dextran-positive neurons were found to be located in the basal plate, close to the pial surface of the hindbrain (Figs. 2A and 2B). VM neurons lay adjacent to a group of Phox2a-expressing cells that were not dextran-labeled and which are likely to be reticular neurons (Tiveron et al., 1996). Thus, facial VM neurons, like all other visceral motor neurons examined so far, express Phox2a. Generation of a Phox2a tau-LacZ Mouse Line We constructed a mouse line in which a tau-lacZ transgene was knocked into the Phox2a locus, creating a fusion between a bifunctional tau-LacZ protein and the NH 2-terminal amino acids of Phox2a (Fig. 3). Correctlytargeted ES cells were identified by PCR analysis, confirmed by Southern analysis, and injected into C57BL/6 host blastocysts to generate chimeric mice. Germ-line transmission was obtained for one clone, and two lines of mice hemizygous for the Phox2a-tau-LacZ allele were generated. These mice did not show any obvious phenotype and were fertile. Intercrosses of heterozygotes produced a Mendelian proportion of homozygous mutant embryos at all stages examined. The embryonic

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FIG. 2. Expression of Phox2a in neurons of the superior salivatory nucleus. (A and B) Retrograde labeling of facial VM neurons with fluorescein– dextran (green) combined with immunofluorescence detection of Phox2a protein (red), in horizontal sections of the hindbrain at the level of the otic vesicle (not shown), in E12.5 wild-type mouse embryos back-labeled from the geniculate ganglion (A) and in E13.5 wild-type mouse embryos back-labeled from the greater superficial petrosal nerve (B). Phox2a is strongly expressed at both stages in VM neurons (white arrows) and in non-dextran-labeled cells (yellow arrows) that are intermingled with motor neurons (see text). Motor axon tracts are indicated by arrowheads in (A). The percentage of Phox2a ⫹ VM neurons (dextran-labeled) in this region as a proportion of the total number of Phox2a ⫹ cells varied between 25 and 54%, with a mean of 38%, in counts from six nonadjacent sections in three hindbrains. The vast majority of dextran-labeled cells were also Phox2a ⫹ (92–100%). In both A and B medial is to the left and dorsal is to the top. Abbreviation: fvm, facial VM neurons. Scale bar in (B) corresponds to 25 ␮m.

phenotype of Phox2a tau-LacZ/tau-lacZ embryos was indistinguishable from that of the Phox2a ⫺/⫺ mutant previously described by us (Morin et al., 1997). Specifically, dopamine-␤-hydroxlase in situ hybridization performed on

homozygous mutants showed that the locus coeruleus was absent; histological analysis at E13.5 showed that the trigeminal, geniculate, and petrosal cranial sensory ganglia were atrophic and that the sphenopalatine gan-

FIG. 3. Targeted disruption of the Phox2a gene. (A) Targeting scheme. (Top) Genomic organization of the Phox2a locus with exons in black. (Middle) Structure of the targeting vector. (Bottom) Predicted structure of the mutant allele before (upper schematic) and after (lower schematic) excision of the neo gene with Cre recombinase. Restriction enzymes: RV, EcoRV; S, SalI. ATG, initiating codon. (B) PCR genotyping of progeny resulting from the mating of hemizygous parents. Homozygous mutants were identified by a band amplified from DNA of embryos 2, 3, and 6 using a pair of LacZ primers and absence of amplification (asterisks) when using Phox2a primers.

18 glion was missing. Finally, other cranial parasympathetic ganglia, such as the otic and submandibular ganglia, showed an incompletely penetrant atrophy (data not shown). In order to visualize the projections of cranial motor axons, whole mounts of E13.5 hemizygous embryos were stained with X-Gal (Fig. 4A). Staining was observed in the geniculate ganglion, the otic ganglion, and the sphenopalatine ganglion and also in the intermediate nerve and the motor root of the facial nerve. The Gspn branches off at approximately 180° to the facial nerve and was readily traced from its origin at the geniculate ganglion to the sphenopalatine ganglion. Staining was also observed in the proximal portion of the facial nerve but was absent in the distal portion of the facial nerve beyond the branch point with the chorda tympani. Therefore, tau-LacZ expression was confined to the VM component of the facial nerve, consistent with our observation that Phox2a is downregulated in facial BM neurons by this stage (data not shown). We next examined homozygous mutant embryos for the presence of the Gspn. At E13.5 we observed a severe abnormality of the Gspn in 7/7 homozygous embryos (14 sides; Fig. 4B). X-Gal staining revealed that the nerve was truncated close to its origin at the geniculate ganglion. Moreover, the remnant of the nerve was oriented at approximately 120° with respect to the facial nerve instead of the normal 180° (Fig. 4A). The length of the remnant also varied among the homozygous mutants examined, but never exceeded approximately oneeighth of the normal length of the Gspn. Neurofilament immunohistochemistry confirmed the abnormality of the Gspn, showing a stump that protruded from the geniculate ganglion and was defasciculated at its distal end (n ⫽ 5 embryos, 10 sides; Figs. 4C and 4D). In contrast, the morphology of the chorda tympani, which also contains VM axons, was invariably normal (data not shown). The presence of the chorda tympani correlated with the persistence of the submandibular ganglion, albeit variably atrophic, in the vast majority of homozygous mutant embryos (data not shown). The morphology of the facial nerve was also normal in these embryos. The absence of the Gspn in the mutants prompted us to verify directly that this was not due to the absence of facial VM neurons. This seemed unlikely given that all BM and VM neurons examined so far are spared by the Phox2a mutation (Morin et al., 1997), probably because their survival and differentiation depend on the early expression of Phox2b (Pattyn et al., 1997, and submitted for publication). However, the overlap of facial VM neurons with the reticular formation has so far pre-

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vented a formal demonstration that these neurons are spared by the Phox2a mutation. To confirm the presence of facial VM neurons in Phox2a homozygous mutants, we injected DiI into the geniculate ganglia of fixed E13.5 embryos (n ⫽ 5). Retrogradely-labeled VM neurons formed a superior salivatory nucleus that appeared to be of normal size and lay in the correct hindbrain location compared to control embryos (n ⫽ 4; Figs. 4G and 4H). Thus, the absence of the Gspn can be attributed to a failure of facial VM neurons to form their usual peripheral projection, rather than to their absence or early degeneration. We next sought to determine whether these neurons projected their axons along the appropriate dorsal pathway. Neurofilament immunohistochemistry on mutant embryos demonstrated an intact intermediate nerve and a normal facial motor root (Figs. 4E and 4F), thereby confirming that BM and VM axons in null mutant embryos leave the hindbrain correctly via a dorsal exit point. Detection of an Ectopic Nerve in E16.5 Null Mutant Embryos Next, we investigated the possibility that facial VM axonal outgrowth toward the site where the sphenopalatine ganglion should form was retarded rather than blocked. We first analyzed heterozygous embryos at E16.5 and observed several important differences from younger controls (compare Fig. 5A with Fig. 4A). All the structures that were visible at E13.5 were more intensely stained in these older embryos, with one exception. Staining in the sensory neurons of the geniculate ganglion was virtually abolished, in keeping with the previous finding of a down-regulation of Phox2a expression between E13.5 and E16.5 (Tiveron et al., 1996). Thus the geniculate ganglion was visible only by virtue of the maintenance of tau-LacZ expression in the preganglionic fibers coursing through it. The otic ganglion was conspicuously enlarged compared to its dimensions at E13.5 and postganglionic fibers could be seen projecting from it. Axonal projections from the sphenopalatine ganglion were longer and more numerous in comparison to younger controls. To determine whether there was a late rescue of Gspn development, homozygous mutants at the same developmental stage were stained with X-Gal. In the region of normal outgrowth of the Gspn we observed a stump of variable length (Fig. 5B), showing that there was no late rescue of the axonal outgrowth defect. An additional anomaly was detected in homozygous mutants at this stage. In five mutants an otic ganglion was present in 7 of the 10 sides. In all 7 cases X-Gal staining revealed a conspicuous connection between

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FIG. 4. Absence of the Gspn and persistence of the superior salivatory nucleus in Phox2a ⫺/⫺ mutants. (A–F) Whole-mount views (A–D) and parasagittal sections (E, F) of E13.5 hemizygous (A, C, E) mouse embryos and homozygous mutant (B, D, F) embryos. (A, B) Dorsal views of the heads of X-Gal-stained embryos, after removal of the brain. (A) In controls the geniculate, otic, and sphenopalatine ganglia are labeled, as are the intermediate nerve, facial motor root, and proximal part of the facial nerve itself. The Gspn can be traced from the geniculate ganglion to the sphenopalatine ganglion. The large out-of-focus and deeply stained structure inferior and to the left of the geniculate ganglion corresponds to the superior cervical ganglion. (B) Representative homozygous embryo showing absence of the Gspn, which is replaced by a stump (asterisk) that protrudes from the geniculate ganglion. The superior cervical ganglion was partially torn during dissection and appears smaller in comparison to its hemizygous equivalent. Differentiating inner ear structures (black arrows in A, B) express endogenous ␤-galactosidase activity. (C, D) Lateral views of whole-mount neurofilament-stained embryos at E13.5 showing a normal Gspn in controls (C), and its absence in homozygous mutants (D), in which a defasciculated stump (arrowhead) is found. (E, F) Parasagittal cryosections of hemizygous control (E) and

20 the geniculate and the otic ganglion (Figs. 5D and 5E). This branch may correspond with the lesser superficial petrosal nerve, which has been documented in humans (Testut and Latarget, 1971) as a group of facial VM axons that innervate the otic ganglion. There is no published evidence thus far of the existence of this nerve in rodents. However, when we examined hemizygous embryos, a thin connection linking the otic ganglion and the geniculate ganglion was visible in some embryos, which in all cases was at the limit of detectability (Fig. 5C). In view of the increased frequency of occurrence of this nerve in null mutant embryos, our favored interpretation is that in the absence of the sphenopalatine ganglion, facial VM axons pathfind to the nearest available alternative parasympathetic ganglion. Sympathetic Axon Pathfinding Is Dependent on the Presence of an Intact Greater Superficial Petrosal Nerve Approximately halfway along its course to the sphenopalatine ganglion, the Gspn is joined by rostrally projecting sympathetic axons from the superior cervical ganglion, forming the vidian nerve (Fig. 5H). Only the parasympathetic fibers synapse on sphenopalatine ganglion neurons, while the sympathetic axons course through and eventually ramify to supply the branches of the external carotid artery. Phox2a is expressed in sympathetic ganglia, and the thick axonal bundles emerging from the superior cervical ganglion and projecting through the sphenopalatine ganglion were readily visualized in Phox2a ⫹/tau-LacZ embryos at E16.5 (Figs. 5A and 5F). In E16.5 homozygous mutants (Figs. 5B and 5G), X-Gal staining revealed that the sympathetic axons stalled at the point at which they would normally fasciculate with the Gspn and had advanced no further rostrally than at E13.5 (Figs. 5H and 5I). Therefore, in the absence of the sphenopalatine ganglion and the Gspn, the sympathetic innervation of the head is truncated. Postganglionic Mash1 Expression Is Dispensable for Preganglionic Axon Pathfinding Mash1 is a bHLH transcription factor that is important for autonomic neurogenesis (Guillemot et al., 1993).

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It is expressed in the sphenopalatine ganglion, raising the possibility that it might regulate putative VM axon guidance cues emanating from this structure and thereby influence facial VM axon pathfinding. In the ventral neural tube, Mash1 expression overlaps the territory in which VM neurons are generated (A. Pattyn, pers. comm.). To begin to investigate the genetic basis of the role of the sphenopalatine ganglion in guiding VM axons, we made use of Mash1 knockout embryos (Guillemot et al., 1993). In these mutants, the sphenopalatine ganglion initially forms and is morphologically indistinguishable from wild type, but degenerates between E13.5 and E16.5. However, cranial motor neurons appear normal. To visualize the axonal pathways in a Mash1 ⫺/⫺ background we crossed males heterozygous for Mash1 with females hemizygous for the tau-LacZ fusion gene. The resulting F 1 double heterozygotes were intercrossed to generate mice with one copy of the tau-LacZ gene in a Mash1 ⫺/⫺ background. F 2 embryos with this genotype were obtained at a frequency approximating the Mendelian proportion of 1/8. We reported previously that the anlage of the sphenopalatine ganglion in Mash1 ⫺/⫺ mice does not express detectable levels of Phox2a (Hirsch et al., 1998). This was puzzling since Phox2a is itself required for sphenopalatine ganglion formation (Morin et al., 1997, and this study). To our surprise, the sphenopalatine ganglion of Mash1 ⫺/⫺; Phox2a ⫹/tau-LacZ mice was readily stained by X-Gal (Fig. 6B). This suggested that Phox2a expression does occur in the sphenopalatine ganglia of Mash1 ⫺/⫺ mice, albeit at greatly reduced levels, and that these low levels of Phox2a are sufficient for initial ganglion formation. In contrast, the expression of Phox2a by cranial motor neurons is not affected in Mash1 mutants (Hirsch et al., 1998, and data not shown). The persistence of the sphenopalatine ganglion at E13.5–E16.5 in Mash1 ⫺/⫺ embryos provided a time window during which to assess the dependence of preganglionic innervation on Mash1 expression in the sphenopalatine ganglion. However, the growth of both Mash1 ⫹/⫺ and Mash1 ⫺/⫺ embryos appeared retarded so that all subsequent analyses were performed at E14.5 rather than E13.5. We found that Gspn development was completely normal in E14.5 null mutant embryos (n ⫽ 2 embryos, 4 sides; Figs. 6A and 6B). Furthermore,

homozygous mutant (F) neurofilament-stained embryos at E13.5 showing the presence of an intact intermediate nerve and facial motor root in homozygous mutant embryos. The trigeminal ganglion is partially visible in both images. (G, H) Flat mounts of hindbrains in E13.5 control (G) and homozygous mutant (H) embryos which were injected with DiI in the geniculate ganglia. Superior salivatory neurons (right arrowhead in H) are labeled as are neurons of the facial motor nucleus (left arrowhead) and their axon tracts (arrow) in controls and mutants. The floor plate lies to the left in both images (not shown). Og, otic ganglion; other abbreviations as in Fig. 1. Scale bar in (G) corresponds to 100 ␮m in (G and H).

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DiI injection into the geniculate ganglia of Mash1 ⫺/⫺ (n ⫽ 5) embryos confirmed the presence of the superior salivatory nucleus (Figs. 6C and 6D). These data indicate that growth of the Gspn is independent of Mash1 expression by the sphenopalatine ganglion.

DISCUSSION We have observed striking axon guidance phenotypes in mice which lack the sphenopalatine ganglion due to a targeted mutation of Phox2a. The navigation of facial VM neurons was disrupted, consistent with a role of the sphenopalatine ganglion in axon pathfinding. In the mutants, VM axons exited the hindbrain normally and navigated as far as the geniculate ganglion, but projected no further (Fig. 7). Analysis at later stages showed that axon pathfinding along this route was not restored. Instead, an ectopic communicating branch linked the geniculate ganglion with the otic ganglion, a target of preganglionic glossopharyngeal VM neurons. In addition, sympathetic axons from the Scg stalled at the point at which they would normally join the Gspn en route to the sphenopalatine ganglion (Fig. 7). The VM pathway formed correctly in Mash1 mutants, sug-

FIG. 5. Parasympathetic and sympathetic nerve abnormalities in E16.5 Phox2a tau-LacZ/tau-LacZ embryos. (A–G) Dorsal views (A–E) and lateral views (F, G) of the heads of X-Gal-stained hemizygous (A, C, F) and homozygous mutant (B, D, E, G) embryos, after removal of the brain, at E16.5. (A) In hemizygous embryos, the Gspn can be seen coursing medially and then rostrally, after having been joined by

sympathetic fibers from the superior cervical ganglion to form the vidian nerve. At this stage, cells in the geniculate ganglion are no longer detectably labeled, and the ganglion is visualized owing to the VM fibers coursing through it. (B) In homozygous mutant embryos, the sphenopalatine ganglion is absent or reduced to a few clusters of cells (black arrowheads). The Gspn is replaced by a stump of variable length (asterisk). (C–E) Higher magnification views of the geniculate and otic ganglia in hemizygous (C) and homozygous mutants (D, E). In occasional hemizygous embryos, such as the one shown in (C), a thin, barely visible communicating branch, the lesser superficial petrosal nerve (arrow), connects the geniculate ganglion to the otic ganglion. (D, E) Two representative examples of homozygous embryos, in which a conspicuous connection is seen between the geniculate ganglion and the otic ganglion (arrows) when the latter is present. In some of these cases, an additional nerve was seen projecting from the otic ganglion (arrowhead). (F, G) Sympathetic axons have fasciculated with the Gspn to form the vidian nerve in control (F) embryos, but in homozygous mutants (G) (see also B) these axons (arrows in B and G) have failed to extend rostrally beyond the point they reached at E13.5 (H, I). The ectopic nerve emanating from the otic ganglion (arrowhead) in (E) is shown also in (G). The lateral view shows that it does not fasciculate with the sympathetic fibers. (H, I) Dorsal view of E13.5 hemizygous control (H) embryo shows rostrally directed sympathetic axons (arrowheads) which are also present in homozygous mutant (I) embryos (arrow). Abbreviations: Sig, sublingual ganglion; Smg, submandibular ganglion; Og, otic ganglion. Other abbreviations as in Fig. 1.

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FIG. 6. Normal outgrowth of the Gspn and vidian nerve in Mash1⫺/⫺ Phox2a⫹/taulac2 embryos. (A, B) Dorsal views of E14.5 hemizygous (A) and homozygous mutant (B) embryos following removal of the brain and X-Gal staining. (A) The Gspn and vidian nerves are clearly seen in the control embryo. (B) Persistence of the Gspn and vidian nerves in null mutant embryos. Note that the contrast has been increased to visualize the Gspn, masking the fact that X-Gal staining in the sphenopalatine ganglion (Spg) is much weaker than in control embryos. (C, D) Flat mounts of hindbrains in E13.5 control (C) and homozygous mutant (D) embryos which were injected with DiI in the geniculate ganglia. The superior salivatory nucleus and the facial motor nucleus (upper and lower arrowheads in D, respectively) are clearly back-labeled in the null mutant. The floor plate lies to the left in both (not shown). Scale bar in (C) corresponds to 100 ␮m in (C and D). Abbreviations as in Fig. 1.

gesting that axon guidance cues are not regulated by Mash1 expression in the sphenopalatine ganglion. Abnormal Facial VM Axon Pathfinding in Phox2a tau-lacZ/tau-LacZ Embryos Our findings in Phox2a tau-LacZ/tau-LacZ mutants indicate that VM axons normally destined to form the Gspn exited the hindbrain in the intermediate nerve and extended toward their intermediate target, the geniculate ganglion. Thereafter, axons stalled distal to the geniculate ganglion, forming a short stump (Fig. 7B). This

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phenotype appeared closely similar in mutant embryos at E13.5 and at E16.5, suggesting that there is no late rescue of nerve outgrowth (Fig. 7D). The observation that interconnected neurons of visceral reflex circuits express Phox2 genes led to the hypothesis that axon pathfinding requires Phox2 expression in neurons and/or their targets (Tiveron et al., 1996; Pattyn et al., 1997). In the present study the pathfinding defects observed could be explained either by the loss of the sphenopalatine ganglion or by the loss of Phox2a expression in the VM neurons. While we cannot unequivocally distinguish between these possibilities, one piece of evidence does point to the loss of Phox2a expression in the Spg as the important factor. In the mutants there is normal outgrowth of chorda tympani axons when their submandibular ganglion target is present, and truncated outgrowth of Gspn in the absence of their target (the Spg). Since Phox2a function has been lost in both neuronal groups this suggests a noncell-autonomous (target-mediated) rather than a cellautonomous function of Phox2a in axon guidance. Further experiments will be required to test the role of Phox2a and Phox2b in axon guidance in more detail. If the target does influence VM axon pathfinding, the ganglion might be the direct source of diffusible axonguidance molecules or might induce its adjacent mesenchyme to produce guidance cues which act on Gspn axons. A large body of evidence now indicates that diffusible chemoattractants play a crucial role in axon navigation (reviewed in Tessier-Lavigne and Goodman, 1996). Among the factors capable of chemoattracting peripheral axons are growth factors, including HGF (Ebens et al., 1996; O’Connor and Tessier-Lavigne, 1999). Candidate guidance and/or survival factors for facial VM neurons may be GDNF and/or its family members, which promote the growth of autonomic neurons and cranial motor neurons (Henderson et al., 1994; Rosenthal, 1999) although no chemoattractant effect has been demonstrated. Although this study suggests that the sphenopalatine ganglion is necessary for facial VM axon pathfinding, the existence of a second signal is suggested by the presence of a stump that branches from the geniculate ganglion in Phox2a ⫺/⫺ mutants. The branching of VM axons at this point occurs despite the absence of the sphenopalatine ganglion in these embryos. In the chick limb bud, axonal defasciculation and sorting are accompanied by the addition of polysialic acid residues to NCAM on axons, which reduces NCAM-mediated adhesion and allows growth cones to respond to specific guidance cues (Rutishauser and Landmesser, 1991; Tang et al., 1994). Genetic analysis of the beaten path

Cranial Visceral Motor Axon Pathfinding

23

FIG. 7. Summary of abnormalities in the patterning of cranial autonomic projections in the periphery in Phox2a mutant mice. (A and B) E13.5. (C and D) E16.5. (A) Hemizygous control embryo. (B) Null homozygous embryo with a stump (arrow) representing the remnant of the Gspn. (C) Pathways in a hemizygous control embryo. (D) Rerouting of facial VM fibers destined for the Gspn to the otic ganglion (arrow) and stalling of sympathetic axons. Abnormal projections in the mutant are indicated in black. In view of the partial atrophy of the submandibular and otic ganglia in null mutants, they are represented by smaller circles in B and D. Abbreviations as in Fig. 1.

gene in Drosophila suggests that selective axon defasciculation at choice points is separable from growth cone steering decisions (Fambrough and Goodman, 1996). Thus, the absence of the target may have unmasked a second, independent component of facial VM axon pathfinding that normally cooperates with targetdependent steering events.

Facial VM Axons Project to Other Ganglia in the Absence of the Sphenopalatine Ganglion In a minority of Phox2a tau-LacZ/tau-LacZ mutant embryos, the otic ganglion was absent, while in all remaining mutant embryos the otic ganglion showed a variable reduction in size. In 100% of these latter cases we detected an axonal projection extending from the geniculate ganglion to the otic ganglion at E16.5. By E16.5 tau-LacZ expression in geniculate ganglion sensory neurons is virtually extinguished. Therefore, the formation of this nerve is presum-

ably due to rerouting of facial VM axons normally destined for the sphenopalatine ganglion (Fig. 7). As sensory axons no longer express lacZ at this stage we could not ascertain their contribution to this nerve. Unequivocal demonstration that this projection consists of facial VM fibers would require retrograde labeling of the otic ganglion in mutant embryos. However, the small size and inaccessibility of the otic ganglion at these embryonic ages precluded this experiment. The navigation of facial VM axons to form the chorda tympani was unaffected in the Phox2a mutant. This raises the possibility that VM axons normally destined to project via the Gspn might instead project along the chorda tympani. It is known that VM neurons that give rise to the chorda tympani lie in a position dorsal to those forming the Gspn (Bruce et al., 1997). However, we did not label these neuronal populations separately and therefore cannot judge whether there was a covert axonal rerouting of the ventral subset of facial VM neurons into other pathways.

24 Stalling Phenotype of Superior Cervical Ganglion Sympathetic Axons in Phox2a tau-LacZ/tau-LacZ Embryos In homozygous mutant embryos, there was a premature termination of postganglionic sympathetic fibers from the superior cervical ganglion at the point where the sympathetic axons normally join the established Gspn pathway to form the vidian nerve. The highly specific and invariant nature of this stalling phenotype suggests that sympathetic axons use the Gspn as a “highway” for their own extension (Tessier-Lavigne and Goodman, 1996) rather than responding directly to cues emanating from the sphenopalatine ganglion. Although it is not possible to rule out a cell-autonomous requirement for Phox2a within Scg neurons, there is no depletion of numbers of these neurons or obvious defects in their differentiation in Phox2a ⫺/⫺ embryos (Morin et al., 1997). Furthermore, in Phox2a tau-LacZ/tau-LacZ embryos the sympathetic chain is intact and showed no obvious defect in axonal projections caudal to the superior cervical ganglion (Morin et al., 1997, and data not shown). Therefore, the sympathetic axonal abnormality is more likely to result from a non-cell autonomous effect of the absence of the Gspn.

Cranial VM Axon Pathfinding Differs from the Pathfinding of Spinal Cord Sympathetic Axons Our study suggests that the ganglionic target of hindbrain parasympathetic VM neurons may have an important role in VM axon pathfinding. Sympathetic VM axon pathfinding in the chick is, in contrast, a targetindependent event (Yip, 1987). Instead, axon guidance cues derived from somitic mesoderm may dictate the route followed by preganglionic sympathetic VM axons (Yip, 1990, 1996). In part, this difference in axon pathfinding characteristics may reflect the distinct ontogenetic histories of these two “visceral” populations. Sympathetic VM neurons in the spinal cord migrate dorsally from their origin among the ventral motor neuron progenitors (Ensini et al., 1998) yet maintain a ventral axonal trajectory as they exit the neural tube, in common with somatic motor neurons. In contrast, parasympathetic VM neurons of the brain stem extend axons via dorsal exit points. Interestingly, ventrally-projecting motor neuron subtypes of the spinal cord, including spinal VM neurons, arise from a population of progenitors expressing a repertoire of transcription factors distinct from those expressed by dorsally projecting hindbrain VM progenitors (reviewed in Pfaff and Kintner, 1998). To distinguish whether these differences between sympathetic and parasympathetic neuron development underlie their different functional specialization and/or

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axon pathfinding it would be useful to determine whether sacral parasympathetic neurons in the spinal cord, which also project in the ventral root (see for example Mawe et al., 1986), require their target for accurate axon pathfinding.

EXPERIMENTAL METHODS Construction of the Phox2a-tau-LacZ Vector A genomic fragment of approximately 14.5 kb containing the entire Phox2a gene and starting 1.7 kb upstream of the start codon was isolated from a mouse 129-SVE phage genomic library. To generate the targeting construct we first isolated an NcoI–XbaI fragment from pETL (Mombaerts et al., 1996) containing the tauLacZ fusion gene. This was introduced into the NcoI and NarI sites of the first exon of Phox2a. A neo gene flanked by two loxP sites (kind gift from H. Gu and K. Rajewsky) was inserted 3⬘ of the tau-LacZ insert. A 10.3-kb fragment of genomic DNA extending downstream of the first exon linked with the HSV-tk cassette at the 3⬘ end constituted the long arm. The short arm extended 1.7 kb upstream of the start codon. ES Cell Culture and Southern Blot Screening of Recombinant Clones ES cell line E14-1 was cultured using standard procedures as previously described (Morin et al., 1997). DNA of recombinant clones was digested by Southern blot with a 0.4-kb EcoRV–EcoRI Phox2a probe located upstream of the targeting construct. This probe hybridizes with a 9.9-kb fragment of wild-type DNA and a 4.7-kb fragment of correctly targeted DNA. Generation of Chimeras and Mating of Mice Chimeras were obtained as previously described (Morin et al., 1997). Germ-line transmission was obtained for one of three injected ES cell lines. Heterozygous male offspring were bred to Cre/Cre females to excise the neo gene. F 2 hemizygous animals were interbred to produce mice homozygous for the tau-LacZ transgene. The animals were genotyped by PCR analysis from tail DNA using two pairs of primers: one pair located within the LacZ gene (5⬘-TCGAGCTGGGGAATAAGCGTTGGC-3⬘; 5⬘-CAAGACCAACTGGTAATGGTAGCG-3⬘) revealed an 814-bp band and another pair consisting of one primer located in the first exon (5⬘-CCTCGGGCCGATGGACTACTC-3⬘) and one primer

25

Cranial Visceral Motor Axon Pathfinding

located in the first intron (5⬘-GGATGGGACTGCGCTCACCTG-3⬘) revealed a 245-bp band.

(peroxidase-conjugated goat anti-mouse; Jackson ImmunoResearch).

Generation of Mash1 ⴚ/ⴚ; Phox2a ⴙ/tau-LacZ Mice

X-Gal Staining

Mice heterozygous for the Mash1 mutation and hemizygous for the tau-LacZ transgene were obtained by breeding Mash1 ⫹/ ⫺ males with females hemizygous for the tau-LacZ transgene. Double heterozygous offspring were interbred to produce Mash1 ⫺/⫺; Phox2a ⫹/tau-LacZ mice. These mice were genotyped using the specific primers described previously (Blaugrund et al., 1996). DiI Labeling DiI crystals (Molecular Probes, Eugene, OR) were dissolved in ethanol at a concentration of 3 mg/ml and injected into the geniculate ganglia of mouse embryos using a nanoliter injector (World Precision Instruments, Sarasota, FL). To ensure adequate uptake embryos were stored in 3.5% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature, in the dark for up to 10 days. The hindbrains were then dissected, flat mounted, and viewed under a confocal microscope (Bio-Rad). Dextran Labeling Labeling with fluorescent dextrans (Molecular Probes) was performed as published (Varela-Echavarrı´a et al., 1996). Embryos were incubated in preoxygenated Hanks’ Basal Salt Solution following dextran labeling. Immunohistochemistry Cryosectioned embryos were washed briefly with PBS and overlaid with 0.1% Triton in PBS (PBSTr) with 20% heat-inactivated sheep serum for 30 min, followed by anti-Phox2a antibody (1 in 200 final dilution) or 2H3 antibody (1 in 1000 final dilution; Developmental Hybridoma Bank, Iowa) in PBSTr and 5% sheep serum overnight. Then further washes in PBSTr/5% sheep serum were performed, prior to incubation with secondary antibodies (Cy3- or FITC-conjugated goat antirabbit or goat anti-mouse antibodies; Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were washed in PBS and mounted in DABCO/glycerol. Sections were viewed under a confocal microscope. Whole-mount immunostaining using 2H3 primary antibody was performed as described (Maina et al., 1997), except that phosphate-buffered saline was used in place of Tris-buffered saline and incubation times were 3– 4 days for primary and secondary antibodies

This was performed as described by Knittel et al. (1995). Embryos were then dehydrated in ethanol and cleared in benzyl alcohol/benzyl benzoate (50/50) for photography.

ACKNOWLEDGMENTS We thank M.-R. Hirsch for help with genotyping and F. Guillemot for the generous gift of the Mash1 knockout mice. We are indebted to Drs. N. Adams for help and advice on DiI labeling, P. Ku¨ry for help with dextran labeling, A. Varela-Echavarrı´a for stimulating discussion and advice on immunohistochemistry, P. Lemaire for the loan of equipment, and J. Livet and C. Barrett for technical assistance. We are grateful to C. Goridis for ongoing discussion and comments on the manuscript and to A. Lumsden and Y. Zhu for comments on the manuscript. This work was funded by an award from the Special Trustees of Guy’s Hospital and the Wellcome Trust and institutional grants from CNRS and Universite´ de Me´diterrane´e. J.J. is a Wellcome Trust Clinical Training Fellow.

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