Target pioneering and early morphology of the murine chorda tympani

J. Anat. (1998) 192, pp. 91–98, with 3 figures Printed in the United Kingdom 91

Target pioneering and early morphology of the murine chorda

tympani

LISA SCOTT AND MARTIN E. ATKINSON

Department of Biomedical Science, University of Sheffield, UK

(Accepted 30 September 1997)

Many studies demonstrate that differentiation of certain sensory receptors during development is induced by

their nerve supply. Thus the navigational accuracy of pioneering fibres to their targets is crucial to this

process. The special gustatory elements of the facial and glossopharyngeal nerves are used extensively as

model systems in this field. We examined the chorda tympani, the gustatory component of the facial nerve,

to determine the precise time course of its development in mice. The transganglionic fluorescent tracer DiI

was injected into the anterior aspect of the mandibular arch of fixed embryos aged between 30 and 50

somites (E10–E12). It was allowed to diffuse retrogradely via the geniculate ganglion to the brainstem for

4 wk, before the distribution of DiI was determined using confocal laser scanning microscopy. Geniculate

ganglion cells were first labelled at the 34 somite stage (E10). Pioneering chorda tympani fibres that arise

from these cells passed peripherally and followed an oblique course as they grew towards the mandibular

arch. At the 36 somite stage (E10.5), the peripheral component followed an intricate postspiracular course

and passed anteriorly to arch over the primitive tympanic cavity, en route to the lingual epithelium. From

the 36 to 50 somite stages (E10±5–E12), it consistently traced in the fashion of a ‘U ’ bend. The central

fascicle also traced at the 36 somite stage (E10±5) and just made contact with the brainstem. At the 40

somite stage (E11), the central fibres clearly chose a route of descent into the spinal trigeminal tract and

branched into the solitary tract. Pioneering chorda tympani fibres contact the lingual epithelium when the

target is primordial. The lingual epithelium may be a source of a neurotropic factor that attracts peripheral

chorda tympani fibres to the sites of putative papillae. However, the chorda tympani is probably not a vital

influence on the subsequent differentiation of gustatory papillae, since the papillae are elaborated 5 d later at

E15 in murine embryos. The early morphology of the nerve is true to the amniote vertebrate phenotype.

Key words : Facial nerve; gustatory papillae.

The first pioneer axons to approach their correct

targets appear to know their home address during

early phases of axogenesis. Some pioneers traverse

considerable distances, coursing through various

substrates and bypass several organs en route, but

remain committed to their natural target.

Soluble factors released by neuronal target tissues

are involved in the formation of normal neural

networks. For example, in the trigeminal system,

explants of placode-derived trigeminal ganglion

neurons of the chick produce neurites in the presence

Correspondence to Dr Martin Atkinson, Department of Biomedical Science, University of Sheffield, Alfred Denny Building, Western Bank,

Sheffield S10 2TN, UK.

of brain-derived neurotrophic factor (BDNF) (Davies

et al. 1986). In the gustatory system, BDNF mRNA is

detectable in both the fungiform and circumvallate

papillae of the developing rat tongue. Its appearance

coincides with lingual epithelium innervation but is

prior to the formation of taste buds (Nosrat & Olson,

1995). BDNF may therefore function as a target-

derived chemoattractant to the special gustatory

neurons.

The navigational fidelity of sensory axons to their

targets is also crucial to the normal development of

certain sensory receptors. For example, the glosso-

pharyngeal nerve induces taste bud formation in rat

1a 1b

Mdc

Bs

Md

VIICTp

Hy

Md

Mx

Op

Fig. 1. (a) Fluorescence microscopy of the murine chorda tympani traced with DiI from the anterior aspect of the mandibular arch of a

fixed mouse embryo at 34 somites (E10). The peripheral axons (CTp) exit the geniculate ganglion (VII) and pass obliquely through the hyoid

arch towards the mandibular arch. Thus the initial axogenesis from the geniculate ganglion extends peripherally and not centrally. DiI also

labelled the mandibular nerve via peripheral axons and the mandibular component of the trigeminal ganglion (Md). The central axons

(MdC) contacted the brainstem (Bs). The close relationship between the mandibular trigeminal ganglion and the geniculate ganglion is

evident. ¬100 (b) Camera lucida drawing of an E10±5 mouse embryo redrawn from Davies & Lumsden (1984) to illustrate the regional

anatomy of the trigeminal and facial nerves. The ophthalmic nerve (Op) and the maxillary (Mx), mandibular (Md) and hyoid (Hy) arches

are shown with their associated cranial nerves. Chorda tympani arrowed. The red box surrounds the region of the head that is captured in

Figure 1a.

posterior lingual epithelium postnatally (Hosley et al.

1987a). Denervation from postnatal d 0 to 10 pro-

duces an impairment of taste bud formation (Hosley

et al. 1987b). Nongustatory vagal afferents are capable

of reinnervating a denervated tongue and restore

normal morphology to the taste buds (Zalewski,

1981). Therefore, the neural induction signal is not

unique to the normal gustatory afferents. Similarly,

the gustatory afferents of the neonatal rat chorda

tympani, which normally innervate taste buds on the

anterior tongue, will also support circumvallate taste

buds on the posterior tongue (Oakley, 1993).

In the fetal rat, there is evidence for an initial

neurotropic effect from the anterior lingual epithelium

(on the chorda tympani and lingual branch of the

trigeminal nerve) and the epithelial cells of the

fungiform papillae apex (on the chorda tympani

only). Initial morphogenesis of the fungiform papillae

commences in the absence of any neural influence but

the rat chorda tympani may induce the differentiation

of taste buds on the papillae (Farbman & Mbiene,

1991). The long-term rabbit neurectomy studies of

Nakashima et al. (1990) demonstrate that once the

receptor-nerve cell relationship is established, the

fungiform papillae become trophically dependent on

the neurons.

Within the trigeminal system of the rat, peripheral

and central axons of the trigeminal ganglion show a

spatial order on reaching the vibrissal field at E12 and

the brainstem at E13 respectively. These events are

prior to the differentiation of the sensory end-organs,

vibrissae follicles and brainstem trigeminal nuclei

(Erzurumlu & Jhaveri, 1992).

The studies outlined above suggest the operation of

92 L. Scott and M. E. Atkinson

Fig. 2. Confocal laser scanning image with a phase-contrast image of the same field overlaid. In this 36 somite specimen (E10±5)

transganglionic tracing of DiI from the anterior aspect of the mandibular arch of a fixed mouse embryo has labelled central (CTc) and

peripheral (CTp) chorda tympani axons and their cell bodies (Cb) in the geniculate ganglion. The morphology of the chorda tympani is

defined by the posterior relation to the spiracle (Sp) and the anterior relation to the tympanic cavity (TC). The spiracular epithelium (Ep)

and the tympanic cavity were discernible using phase-contrast microscopy. DiI also labelled the mandibular component of the trigeminal

ganglion (Md) via the peripheral fascicle (P). ¬160.

a vital intrinsic influence governing the phenotype of

pioneer neurons. Consequently, in particular receptor-

nerve cell systems, such neurons exert profound effects

on the differentiation of their sensory end-organs.

Kuratani et al. (1988) described the morphology of

the chorda tympani during chick development. Using an

immunohistochemical staining technique, they revealed

both prespiracular (stage 17) and postspiracular

(stage 21) components of the chorda tympani, which

together encircled the spiracle (the upper part of the

first branchial groove). This so called ‘spiracular

loop’ appears to be unique to the chick since Goodrich

(1914) demonstrated that the chorda tympani in

amniote vertebrates is entirely postspiracular.

The aim of the present study is to examine the

course of developing peripheral and central chorda

tympani axons and the time that they pioneer their

respective targets. From our experimental data it is

possible to speculate upon the importance of target-

derived chemotropic influences and the inductive

Development of the chorda tympani 93

relationship between axons and their sensory recep-

tors.

Pregnant time-mated MF"mice between 10 and 12 d

of gestation were killed by cervical dislocation. The

presence of a vaginal plug was designated as E0. The

uteri were removed and placed in phosphate buffered

saline (pH 7±2). The embryos were dissected from the

uteri and extraembryonic membranes then accurately

aged by both external morphological criteria and by

somite counts (Theiler, 1989). The embryos were

decapitated immediately and the heads fixed in a

solution of phosphate buffered 4% paraformaldehyde

and 0±2% glutaraldehyde (pH 7±2) for 1 d at room

temperature.

Crystals of the fluorescent carbocyanine neuronal

tracer DiI [1,1«-dioctadecyl-3,3,3«,3«-tetramethylindo-

carbocyanine percholate ; DiI-C18-(3)] (Molecular

Probes Oregon, USA) were dissolved in dimethyl-

formamide (1±5 mg in 200 µl). Small quantities of

solution were drawn up by capillary action into heat

stretched glass micropipettes, which were used to

insert the tracer. DiI was injected unilaterally into the

anterior aspect of the right mandibular arch in 30 to

50 somite stage embryos (E10–E12) to trace the

peripheral and central components of the mandibular

nerve and the chorda tympani to the brainstem. In all

cases, suction of excess fluid out through the stomo-

deum by micropipette capillary action prevented

spread of tracer to other areas.

Successful placement of tracer into the correct

target area was monitored by a primary examination

using an inverted Leitz fluorescence microscope

equipped with TRITC filters. When the correct

location of the tracer was confirmed, the injected

heads were returned to fixative in 2 ml eppendorf

tubes. These were wrapped in aluminium foil to

prevent photobleaching. The tissues were stored at

room temperature in the dark for between 1 and 4 wk

to allow the tracer to diffuse retrogradely.

After 1–4 wk, each head was bisected in the sagittal

plane and the distribution of the tracer in the right

half observed using fluorescence microscopy in com-

bination with phase-contrast microscopy. Each speci-

men was mounted in PBS on a 35 mm tissue culture

dish, with the lateral aspect of the bisected head

placed inferiorly. Photomicrographs were taken on

Kodak Gold 200 colour print film and this was

developed and printed routinely.

Several specimens received more detailed exam-

ination. These were mounted beneath a coverslip in

PBS on a well slide, with the lateral aspect of the

bisected head placed superiorly. Optical sections were

obtained at 2–22 µm intervals to a maximum depth of

176 µm using an upright Leica TCS 4D confocal laser

scanning microscope set up for observation of DiI. A

standard TRITC filter set was used and each specimen

was excited at 568 nm with a 590 nm low pass emission

filter (Honig & Hume, 1989).

Between 8 and 16 full frame images (512 by 512

pixels) were collected and stored for each specimen.

These images were further processed using the

manufacturer’s software so that serial sections were

stacked to give extended focus views. The colour of

the tracer was then assigned electronically to give the

characteristic appearance of red-orange DiI.

Phase-contrast images were obtained on the CLSM

using a detector mounted beneath the condenser set

up with appropriate rings. Fluorescence and phase-

contrast images were overlaid to define the chorda

tympani in relation to the spiracle and the tympanic

cavity. The final images were sized and labelled using

the Micrografx Picture Publisher LE 4.0a programme.

A Lasergraphics slide maker transferred the electronic

images onto Kodak Gold 100 colour print film and

this was developed and printed routinely.

(see Table)

30–33 somites (E10)

There was no labelling of chorda tympani axonal

processes or geniculate ganglion cell bodies between

30 and 33 somite stages. DiI was restricted to the

insertion site, although secondary transcellular label-

ling was detectable in the surrounding mesenchyme

(data not shown).

34–35 somites (10–10±5)

The chorda tympani was first labelled at this stage.

Only the peripheral fascicle traced with DiI from the

mandibular arch to the geniculate ganglion (Fig. 1a).

The fascicle exited from the peripheral aspect of the

ganglion and passed obliquely through the hyoid arch

towards the mandibular arch. Only 2–3 neurons

were present in all speciemens examined. No central

fibres were discernible, which indicates that the initial

axogenesis from the geniculate ganglion extends

peripherally and not centrally.

In addition to the chorda tympani, the mandibular

nerve traced at the 34 somite stage. DiI was

transported retrogradely to the brainstem in per-

ipheral and central axons of bipolar neurons via the

mandibular component of the trigeminal ganglion


(Fig. 1a). Figure 1b illustrates the lateral aspect of an

E10±5 mouse embryo to demonstrate the regional

anatomy of the trigeminal and facial nerves.

36 somites (E10±5)

In addition to the peripheral fascicle, the centrally

oriented fascicle of the chorda tympani (a component

of the nervus intermedius) traced at this stage.

Consequently, the complete first order pathway

composed of bipolar neurons was discernible for the

first time (Fig. 2). This central outgrowth travelled a

short distance from the geniculate ganglion to make a

direct contact with the brainstem.

The peripheral fascicle passed downwards and

beneath the spiracular epithelium to follow a post-

trematic course into the hyoid arch. It passed posterior

and inferior to the spiracle and thence continued

anteriorly and superiorly, arching over the posterior

aspect of the primitive tympanic cavity towards the

mandibular arch, creating a ‘U-bend’ en route. In

all 35–37 somite specimens examined, only 2–8

pioneering chorda tympani neurons were visible.

37–39 somites (E10±5)

The morphology of the chorda tympani continued as

described above except the ‘U-bend’ developed a more

acute curve. The peripheral and central fascicles

became progressively more robust but accurate

neuron counting was no longer possible since the

fibres were intermingled and compacted. The central

fascicle gradually descended further into spinal tri-

geminal tract.

40 somites (E11)

The peripheral axons of the chorda tympani arose

from the peripheral aspect of the geniculate ganglion

and at an approximate right angle to the central

fascicle (Fig. 3a, b). The central fascicle branched so

that fibres distributed to the spinal trigeminal tract

and also to the domain of the tractus solitarius, which

lies dorsal to this tract (Fig. 3a, c). It is likely that the

branch to the tractus solitarius contains the special

visceral afferents for taste. The axons entering the

spinal trigeminal tract are possibly general somatic

afferents of the facial nerve. The latter vary enormously

between individuals and behave like the afferents of

the trigeminal nerve once in the spinal trigeminal tract

(Nolte, 1993).

41–50 somites (E11–E12)

The complete chorda tympani tracing was repro-

ducible without deviation throughout these stages. In

the age ranges examined, the peripheral component of

the chorda tympani did not fasciculate with the

neighbouring mandibular division of the trigeminal

nerve. Figure 1a shows a general view of the

relationship between the mandibular division of the

trigeminal ganglion and the geniculate ganglion, with

the associated axonal processes present at the 34

somite stage (E10). The results are summarised in the

Table.

This in vivo dye tracing study demonstrates that

pioneer peripheral axons in the chorda tympani reach

the lingual epithelium at the 34 somite stage (E10).

This projection is the first outgrowth from the

geniculate ganglion and the central fascicle arises 2

somite stages later. The early morphology of the

chorda tympani corresponds to the characteristic

amniote vertebrate design.

Axons did not trace at the 30–33 somite stages

(E10). This indicates either the absence of axonal

projections from the chorda tympani or that pioneer-

ing neuron had not reached the source of the tracer by

this time. It is therefore possible that pioneering axons

had embarked on their journey to the periphery

during this time but were not traceable in these early

stages. At the 34 somite stage (E10), the peripheral

component of the chorda tympani traced for the first

time. It followed an oblique route to the mandibular

arch, which was relatively direct when compared with

later stages. The subsequent tortuous course of the

nerve appears to be secondary to the dynamic

sculpting of the branchial region of the head and is

not convoluted orienteering. The central component

of the chorda tympani traced 2 somite stages later at

the 36 somite stage (E10±5). The development of the

chorda tympani is truly ‘outside-in ’ since the central

projection arises subsequent to the initial peripheral

axogenesis. This result contrasts the early pioneering

behaviour of the murine trigeminal system in which

the maxillary and mandibular nerves simultaneously

extend axons both peripherally and centrally (Scott &

Atkinson, unpublished). This ‘outside-in ’ sequence

conforms to the development of the gustatory path-

ways at higher levels. For example, dendrites of

neurons in the rat rostral nucleus of the solitary tract

ramify extensively between postnatal d 6 and 20.

During this period the volume of the chorda tympani


Fig. 3. Confocal laser scanning images to show transganglionic tracing of DiI from the anterior aspect of the mandibular arch of a fixed

mouse embryo at 40 somites (E11). (a) The peripheral axons of the chorda tympani (CTp) arise from the geniculate ganglion (VII) at an

approximate right angle to the central axons (CTc). The central fascicle branches so that fibres distribute to the long spinal trigeminal tract

(SpV) and to the domain of the tractus solitarius (Br). ¬160. (b) Higher magnification image of the cell body detail in the geniculate ganglion.

¬250. (c) Higher magnification image of the branch to the tractus solitarius with the zoom option applied. ¬250.

terminal field doubles in the nucleus of the solitary

tract (Lasiter et al. 1989). Furthermore, synapse and

dendrite development in the rat parabrachial gus-

tatory zone at the second order level occurs sim-

ultaneously between postnatal d 18 and 35. This event

follows morphological development in the first order

pathway and the rostral nucleus of the solitary tract

(Lasiter & Kachele, 1988, 1989, 1990).

‘Outside-in ’ development also reflects the trophic

dependence of the central projection on the intact

peripheral projection in adult rodents. For example,

severance of the hamster chorda tympani induces

degeneration of the central terminals in the nucleus of

the solitary tract. This degeneration arises from

altered synaptology and not ganglionic cell death

(Whitehead et al. 1995). Therefore, the finding that

peripheral projections develop prior to their central

counterparts strengthens the evidence that anatomical

integrity and physiological health are crucial to the

maintenance of gustatory competence in adulthood.

Gross innervation of murine lingual mucosa is well

established by E12 but gustatory papillae do not

appear until E15 (Nolte & Martini, 1992) and taste

bud differentiation in circumvallate papillae com-

mences on d 0 after birth (Takeda et al. 1992). The 5 d

time lag between pioneering innervation of lingual

epithelium at E10 and appearance of the papillae

makes it highly unlikely that nerves induce papilla


Table. Summary of results

Age of embryo Retrograde dye tracing*

Embryonic day Somite count CTp CTc Sp Br

E10 30 ® ® ® ®31 ® ® ® ®32 ® ® ® ®33 ® ® ® ®34 ® ® ®

E10±5 35 ® ® ®36 ®37 ®38 ®39 ®

E11 40–49 E12 50

* CTp, peripheral axons of chorda tympani traced from anterior

aspect of mandibular arch to geniculate ganglion. CTc, central

axons of chorda tympani traced transganglionically from anterior

aspect of mandibular arch to contact brainstem. Sp, chorda

tympani passed posterior to spiracle and anterior to tympanic

cavity en route to the mandibular arch. Br, chorda tympani entered

tractus solitarius and some axons progressively descended further

into spinal tract.

formation and differentiation. Furthermore, there is

recent evidence that gustatory papillae develop with-

out neural influence. Organ cultures of embryonic rat

tongue show normal papillary spatiotemporal differ-

entiation (Mbiene et al. 1997). There is also striking

evidence that BDNF is preferentially expressed in

putative gustatory areas of the lingual epithelium

prior to and during papillary differentiation and

formation (Nosrat & Olson, 1995; Nosrat et al. 1996).

BDNF may function as a short range chemotropic

mechanism to delineate innervation of papillae from

subepithelial plexuses, since in BDNF null mutant

mice, taste bud formation and their intraepithelial

innervation is impaired (Nosrat et al. 1997).

The factors that direct the initial axogenesis of

peripheral and central chorda tympani projections

when they arise from the geniculate ganglion remain

elusive. Nevertheless, it is apparent from our study

that an initial chemotropic influence from the murine

target tissue could take effect when the target is in

premature stages of differentiation before the 34

somite stage (E10). Therefore, putative tropic factors

are likely to be regionally attractive and not specifically

attractive to guide axon terminals to sensory end-

organs.

In the present study, the peripheral fascicle adopted

the characteristic morphology of the chorda tympani

at the 36 somite stage (E10.5). The tracing pattern was

replicated up to the 50 somite stage (E12). Kuratani et

al. (1988) presented an anatomical definition of the

chick chorda tympani using its relation to the spiracle

and the tympanic cavity as defining factors. DiI tracing

revealed that the peripheral fascicle of the murine

embryo passes posterior to the spiracle and progresses

downward into the hyoid. It then turns upward

forming a ‘U-bend’ that arches over the posterior

aspect of the tympanic cavity, before continuing

anteriorly to the tongue. The chorda tympani in the

murine embryo is therefore postspiracular. This

contrasts with the arrangement in the chick, in which

the nerve is mostly prespiracular with a less robust

postspiracular branch. Together, these 2 branches

form a loop that encircles the spiracle (Kuratani et al.

1988). The general morphology of the chorda tympani

in the present study is consistent with the fundamental

observation that the chorda tympani always locates

between mandibular and hyoid skeletal elements

(Gaupp, 1912, cited by Kuratani et al. 1988). More

specifically, the murine chorda tympani is entirely

postspiracular, conforming to the more usual ar-

rangement in amniote vertebrates (Goodrich, 1914).

The mandibular nerve and the chorda tympani did

not appear to fasciculate in the periphery at any of the

embryonic stages studied in this investigation. How-

ever, the extreme peripheral courses of the mandibular

nerve and the chorda tympani were masked by the

injection site. Therefore, fasciculation at their extremi-

ties is a possibility. Nevertheless, numerous photo-

micrographs and camera lucida drawings of murine

cranial nerve development in the literature do not

show fasciculation of the mandibular nerve and the

chorda tympani at E10–E11 (see, e.g. Davies &

Lumsden, 1984, fig. 1; Meyer & Birchmeier, 1995, fig.

4).

In conclusion, the murine chorda tympani is

postspiracular and pioneers the lingual epithelium at

the 34 somite stage (E10) via a relatively direct route.

The intricate morphology of the nerve at the 36 somite

stage (E10±5) is not convoluted orienteering but is

secondary to the sculpting of the branchial region of

the head. The undifferentiated lingual epithelium may

be a source of a chemotropic factor that facilitates

chorda tympani navigational success once it has

proceeded distal to the developing outer and middle

ear cavities. Considering that the lingual papillae

develop following the initial innervation of the tongue,

chorda tympani nerve fibres are not dependent on

their sensory end-organs for guidance. Due to the

considerable time-lag between lingual innervation and

papillary differentiation, the nerve supply evidently

does not induce morphological changes in the lingual

epithelium at the early pioneering stage, confirming

the observations of Farbman & Mbiene (1991).


The authors thank Dr Adrian Jowett for assistance

with the operation of the confocal laser scanning

microscope and production of the final images. The

microscope was funded by the Wellcome Trust. This

study was supported by a scholarship awarded to Lisa

Scott by the Anatomical Society of Great Britain and

Ireland.

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Target pioneering and early morphology of the murine chorda tympani