Ž .Developmental Brain Research 105 1998 85–96

Research report

The morphogenesis of mouse vallate gustatory epithelium and taste budsrequires BDNF-dependent taste neurons

Bruce Oakley ), Adam Brandemihl, Dylan Cooper, David Lau, Anne Lawton,Chunxiao Zhang

Department of Biology, UniÕersity of Michigan, Ann Arbor, MI 48l09-1048, USA

Accepted 7 October 1997

Abstract

Ž .The developmental absence of brain-derived neurotrophic factor BDNF in null mutant mice caused three interrelated defects in thevallate gustatory papilla: sparse innervation, a reduction in the area of the gustatory epithelium, and fewer taste buds. On postnatal day 7,the stunted vallate papilla of bdnf null mutant mice was 30% narrower, the trench walls 35% reduced in area, and the taste buds 75% lessabundant compared with wild-type controls. Quantitative assessment of innervation density was carried out to determine if the smalltrench walls and shortage of taste buds could be secondary consequences of the depletion of gustatory neurons. The diminished gustatory

Ž . Ž .innervation was linearly associated with a reduced trench wall area rsq0.94 and fewer taste buds rsq0.96 . Residual taste budswere smaller than normal and were innervated by a few surviving taste neurons. We conclude that BDNF-dependent taste neuronscontribute to the morphogenesis of lingual gustatory epithelia and are necessary for both prenatal and postnatal mammalian taste budformation. The gustatory system provides a conspicuous example of impaired sense organ morphogenesis that is secondary to sensoryneuron depletion by neurotrophin gene null mutation. q 1998 Elsevier Science B.V.

Keywords: Axon; Brain-derived neurotrophic factor; Foliate papilla; Fungiform papilla; Mouse; Neurotrophin-3; Tongue

1. Introduction

Apart from their familiar role in propagating codedmessages, mammalian sensory neurons nurture and sustain

w xseveral types of developing secondary sensory cells 57 .In addition to taste buds, these include muscle spindlesw x w x w x56 , tendon organs 58 , Pacinian corpuscles 59 , and

w xMeissner corpuscles 26,60 . The failure of neurotrophin-3Ž . w xNT3 null mutant mice either to sustain Merkel cells 2 ,or to develop proprioceptive neurons and associated senseorgans, provides further evidence that the development of

w xsecondary sensory cells is nerve-dependent 16,18,31 . In acomplementary manner, transgenic overexpression of NT3causes profuse sensory innervation and enhances skin de-

w xvelopment 3 . The present research focuses on mouse tastebuds and associated gustatory epithelia as specific exam-ples illustrating two non-coding consequences of sensoryneurons: the contributions of sensory innervation to senseorgan development and to skin morphogenesis.

) Corresponding author. Department of Biology, 3127 Natural ScienceBuilding, University of Michigan, Ann Arbor, MI 48109-1048. Fax: q1Ž .313 647-0884; E-mail: boakley@umich.edu

Taste buds on the mouse tongue are located in gustatorypapillae. Over 100 fungiform papillae in loose rostro-caudalrows are deployed over the anterior two-thirds of the adultmouse tongue where they receive both trigeminal andfacial nerve sensory innervation. The posterior third of thetongue has several laterally situated foliate papillae andone vallate papilla. As a result of its caudal midlinelocation, vallate tissue is exclusively and extensively inner-vated by the right and left IXth nerves of the petrosalganglia. In mice one week old, vallate gustatory trenchwalls contain about 100 taste buds while foliate trenchwalls contain dozens of taste buds.

Postnatal neuronal–epithelial interactions in the rat val-late papilla contribute to the gustatory competence of thevallate epithelium, and to the subsequent formation of taste

w xbuds during a sensitive period 24,25,40–42,46 . Althoughvallate taste bud formation is largely postnatal in rat andmouse, it would not be surprising if embryonic innervationalso made critical prenatal contributions to the develop-

w xment of gustatory papillae and taste buds 23,51 . Underthis model, the embryonic demise of taste neurons wouldprevent taste bud development. Alternatively, taste budsmight arise without embryonic innervation, in which case

0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 97 00178-8

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–9686

the well-known nerve-dependence of adult taste buds wouldbe acquired later, as is the nerve-dependence of salaman-

w xder limb regeneration 19,41 .Peripheral taste development in the mammalian fetus

has been studied by Bradley, Mistretta and co-workersusing sheep whose precocious development facilitated lim-

Ž w x.ited surgical interventions in utero reviewed in Ref. 34 .Allied experiments in altricial mammals like mice havebeen stymied by the difficulties of fetal intervention. How-ever, by substituting genetic deletions for surgical dele-tions in the analysis of gustatory innervation, the use oftargeted mutations in mice may bypass the classic road-block of early embryological surgery in mammals. Specifi-cally, null mutant mice that lack the neurotrophin requiredby embryonic taste neurons provide an experimental op-portunity to evaluate the effects of the embryonic depletionof taste neurons on mammalian taste bud developmentw x37,63 .

wFour mammalian neurotrophins nerve growth factorŽ . Ž .NGF , brain-derived neurotrophic factor BDNF , and

Ž .xneurotrophin-3 and -4 NT3 and NT4 function as target-derived survival factors that support a subset of embryonic

Ž w x.neurons reviewed in Ref. 9 . BDNF supports the devel-opment of vestibular, auditory type 2, and ventilatory

w xvisceral afferents 13,16 . Accumulating evidence also im-plicates BDNF in the support of developing taste neurons.In situ hybridization has shown preferential, prenatal andpostnatal associations of BDNF mRNA with lingual gusta-

w xtory papillae 38,39 . The possibility that BDNF acts as asurvival factor for taste neurons can be addressed more

w xdirectly with bdnf null mutations 9,14,27,32 . Recentstudies of null mutant mice indicate that the normal devel-

w xopment of taste neurons in vivo requires BDNF 37,63w xand its receptor, trkB 20 .

We have previously shown a striking loss of gustatoryinnervation in bdnfyry mice and reported further that thevallate gustatory papilla had few taste buds and was

w xstunted 63 . The present study provides quantitative mea-sures sufficient to test for a possible linkage betweensparse gustatory innervation and the ensuing flawed em-bryogenesis of mouse gustatory papillae and taste buds.These findings bolster two biological principles. First,nerve-dependent embryological events are required for thedevelopment of several secondary sense organs like mam-malian taste buds. Second, neurotrophin-dependent sensoryneurons support the morphogenesis of specialized cuta-neous epithelia.

2. Materials and methods

2.1. Tissue collection

Breeding pairs of adult mice heterozygous for a bdnfw xnull mutation 14,32 were obtained from The Jackson

Laboratory, Bar Harbor, ME 04609-1500. All mice in the

breeding colony were kept on a 12-h light:12-h dark cycleand housed with ad libitum food and water. Pups weretypically delivered at night. The following noon was de-fined as postnatal day 0.5. Bdnf null mutant neonatal micegenerally survive for no longer than 2–3 weeks. Neonataloffspring were euthanized with an overdose of an i.p.

Ž y1 .injection of sodium pentobarbital 150 mg kg b.wt orwith carbon dioxide gas. We followed the supplier’s in-structions for PCR-based genotyping of mouse tail DNA toidentify wild-type, heterozygous, and homozygous nullmutant bdnf mice. We made cryostat sections of thetongue and palate of bdnfqrq and bdnfyry mice after

Žfixation in 4% formaldehyde or acid alcohol 10% acetic.acid and 70% ethanol .

In preparation for sectioning, the tissue was cryopro-tected in increasing concentrations of sucrose and frozen ina 2:1 mixture of 20% sucrose and the embedding com-

Ž .pound OCT Miles, Elkhart, IN 46515 . Cryosections of20-mm thickness for nerve staining and 30 mm thicknessfor taste bud staining were cut at y258C, thaw-mountedon gelatin-coated slides, and stored at y208C.

2.2. Immunohistochemical staining of taste buds

Ž .The monoclonal antibody mAb Troma-I readily iden-w xtified taste buds by its specific reaction with keratin 8 50

Ž .Hybridoma Bank, Univ. of Iowa, Iowa City, IA 52242 .Immunohistochemistry was carried out using an avidin–

Žbiotin peroxidase method standard ABC kit, Vector Labs.,.Burlingame, CA 94010 . The mounted acid alcohol fixed

tongue sections were hydrated with four 5-min washes in0.4% Triton X-100 in 0.1 M phosphate buffered salineŽ .PBS , pH 7.4. The slides were next exposed to 3% normal

Žgoat serum NGS; aG-6767, Sigma Chemical, St. Louis,.MO 63178 for 30 min, followed by overnight incubation

at 48C in 1:60 MAb Troma-I in 0.1 M PBS containing0.4% Triton X-100 and 3% NGS. Upon rewarming toroom temperature for 1 h, the slides were washed withPBS four times for 6 min each, followed by a 60-minapplication of the secondary antibody, biotin-conjugated

Žgoat anti-rat IgG 1:1500 in PBS, a112-065-102, Jackson.Immunoresearch Labs, West Grove, PA 19390 . Four addi-

tional washes in PBS preceded both the 30-min applicationŽof the Vectastain avidin–biotin complex PK-4000 kit,

.Vector and the 5–10 min incubation with a PBS solutionX Žcontaining 0.5 mgrml 3,3 -diaminobenzidine DAB,

.Sigma , 0.01% hydrogen peroxide, and 0.04% NiCl to2w xtint the reaction product blue 30 . Positive controls in-

Ž .cluded salivary duct cells for keratin 8 Troma-I . Omis-sion of the primary antibody eliminated all staining.

2.3. Immunohistochemical staining of axons with PGP 9.5

Nerve fibers were stained with rabbit polyclonal anti-Žserum to human protein gene product 9.5 PGP 9.5,

1:800–1:1600; a7863-0504, Biogenesis, Sandown, NH

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–96 87

.03873 . In formaldehyde, fixed tissue anti-PGP 9.5 recog-nizes ubiquitin hydrolase in axoplasm. Formaldehyde fixedtongue sections to be used with anti-PGP 9.5 were hy-drated in one 5 min rinse in 0.1 M PBS, placed for 20 minin 0.3% H O , and then rinsed again three times for 5 min2 2

each in PBS. The slides were next incubated for 30 minwith 3% NGS in 0.1 M PBS with 0.4% Triton X-100,followed by two 4-min washes with PBS. The primary

Ž .antibody anti-PGP 9.5 was diluted 1:800–1:1600 in 0.1M PBS containing 3% NGS and 0.4% Triton X-100, andapplied overnight at 48C. The slides were subsequentlywashed with PBS four times for 4 min each and exposedfor 1 h to the secondary antibody, biotin-conjugated goat

Žanti-rabbit IgG 1:200 dilution in PBS, aSC-2015, Santa.Cruz, Santa Cruz, CA 95060 . Four additional washes in

PBS preceded both the 30-min application of the Vectas-tain avidin–biotin complex and the 5–10 min incubationwith a PBS solution containing 0.5 mgrml DAB, 0.01%hydrogen peroxide, and 0.04% NiCl . PGP 9.5 staining2

provided excellent resolution of the taste axons’ fine termi-nal branches in the gustatory epithelium and taste buds.Positive controls included lingual motor axons for nervefibers. Omission of the primary antibodies eliminated spe-cific staining.

2.4. Measurements of taste bud size

Since taste buds were generally less than 50 mm indiameter, most were encompassed by two 30-mm thicksections. The exact area of the largest section through a

Ž .taste bud its profile was used to characterize the size ofŽvallate taste buds in cross-sections of the tongue we did

not attempt volume estimations that are subject to inaccu-racies since the gustatory tissue does not extend the fullthickness of a section that slices through the margin of a

.taste bud . Taste bud profiles were examined with a 100=

oil-immersion objective, traced with camera lucida, andthe area measured with a digitizing tablet and computer.Comparisons between experimental and control mice weremade without correction for tissue shrinkage.

2.5. Structural measurements of the Õallate

The vallate papilla’s width was defined as the separa-tion between the two trenches at their rostro-caudal mid-point. The width of the vallate and the depth of thetrenches were measured to provide quantitative indices ofpapilla stunting. The shape and area of vallate trench wallswere then reconstructed from trench depth measurementsin complete serial sections at a thickness of either 20 mmor 30 mm. All trench depth measurements were obtainedfrom cross-sections of uniformly mounted tongues. Thetop of the trench opening was referenced to the dorsal

Žsurface of the outer trench walls the fringing epithelium.of the vallate papilla . Had the dorsum of the vallate

papilla been used as the reference point for trench height,it would have accentuated the stunting of the null mutanttrenches, since wild-type mice had a more elevated papilla.Because the 2-D trench wall reconstructions are basedupon trench depth, they exclude the thickness of theepithelium at the base of the trench, and thus represent theshape of the exposed portion of the trench wall. Theposition of each taste bud was established by extending itsprincipal axis to the surface of the trench wall.

2.6. Measurements of innerÕation density

Axon abundance within the vallate papilla or within thetrench walls was ranked as low, medium, or high. Inaddition, taste bud innervation density was assessed bycounting intragemmal varicosities of the largest sectionthrough a taste bud. The innervation density of a taste bud

Žwas defined as the number of varicosities swellings greater.than 1 mm in all directions per unit area of the taste bud

profile. Swollen and dark, varicosities in taste buds couldbe counted with "15% reliability. To quantify innervationdensity, lingual tissue from five bdnfyry mice was pro-cessed under identical conditions. Specifically, each vallatewas serially sectioned at a thickness of 20 mm and stained

Ž .Fig. 1. Transverse sections of P7 mouse vallate papilla were stained with MAb Troma-I. Examples of taste buds solid arrows and salivary duct cellsŽ . Ž . qrqopen arrows are indicated. Salivary duct cells contain keratin 8 and, therefore, are also immunopositive for Troma-I. A The trenches in this P7 bdnfvallate had 92 taste buds, of which more than 20 are visible in this section. Also evident are two of nine taste buds on the dorsal surface of the vallate

Ž . yrypapilla proper. The vertical line shows the shallow depth of the left trench. B This P7 bdnf vallate had a total of only two taste buds; one is visible inŽ . yrythis section. The remaining stained cells are polygonal cells of salivary ducts. The vertical line shows the depth of the left trench. C This P7 bdnf

vallate contained a total of eight taste buds. Light staining with Troma-I reveals the oval nuclei and characteristic elongation of the intragemmal cells inŽ . Ž . Ž .contrast to the polygonal form of salivary duct cells. Scale bar in A is 90 mm for A–B and 20 mm for C .

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–9688

with anti-PGP 9.5 at a dilution of 1:1600. This dilutionwas determined to be optimal for facilitating counting ofvaricosities.

2.7. Assay for taste-mediated drinking behaÕior

For an assay of function, behavioral assessment wasselected over electrophysiological recording because mostbdnfyry mice die before recording is feasible. Further,selective sucrose ingestion provides a more comprehensivedemonstration, since it requires functional integrity of boththe peripheral and the central gustatory connections. Toavoid generating thirst that might override gustatory cuesand lead to indiscriminate fluid ingestion, the neonateswere not fluid-deprived before testing. Each tested litter ofP7, P12, or P14 neonates was taken from its dam andplaced in a holding box at 308C. Within 15–45 min afterseparation from the dam, individual mice were sequentiallyremoved from this box and behaviorally tested. Each mousewas supported upon a raised gloved hand so its mouthcould be clearly visualized. As soon as the mouse wasmotionless, a 15-ml fluid drop of distilled water wasapplied to its lips. About 30-s intervals separated thepresentation of each of the subsequent taste solutions. Thepresentation sequence was: distilled water, 0.3 M sucrose,0.001 M quinine monohydrochloride hydrate, distilled wa-ter, 0.3 M sucrose, and 0.001 M quinine monohydrochlo-ride hydrate. Two observers characterized each of the six

Fig. 2. Histograms of vallate taste bud areas in three P7 bdnfqrq miceŽ .ns248 taste buds randomly sampled from a population of 322 and all

yry Ž40 taste buds in five P7 bdnf mice ranges2–17 vallate taste.budsrmouse . From cross-sections of the tongue, measurements of the

largest section through each vallate taste bud were cast into 200 mm2

wide bins. The mean areas of taste bud profiles are 347 mm2 for bdnfyry

and 614 mm2 for bdnfqrq. The shading of the P7 bdnfyry histogrammasks P7 bdnfqrq data entries of three taste buds in the 0–99 um2 binand seven taste buds in the 100–199 mm2 bin.

Fig. 3. Histological reconstructions of left and right vallate trench wallsare shown for one wild-type and three null mutant mice. Reconstructionsare based upon measurements of trench depth in complete serial sectionsof each vallate. In this sagittal view of the trench walls, anterior is to theleft. Projection of the long axis of each taste bud onto the trench wallsurface established the location of each taste bud in the outer trench wallŽ . Ž .open circles and inner trench wall filled circles . As the developingtrench deepens, more of the ventral taste buds will become exposed. Thescale bar is 200 mm.

responses as either: no response, intake that always tookthe form of stereotyped, repetitive suckling movements, oraversion such as head deviation or retraction. Because few

Žmice 13% of wild-type mice and 15% of null mutant.mice showed aversion to quinine, we present data only on

responses to water and sucrose. The genotype of each

Fig. 4. The number of taste buds per vallate trench wall is a function ofŽ .trench wall area heavy dashed line: r sq0.71 . The straight dotted line

is the theoretical line of perfect proportionality, e.g., with an X interceptof zero. Within each animal, data from its right and left trenches areconnected by a fine line. P7 bdnfyry, ns7 mice; P12 bdnfyry, ns5mice. Taste buds were counted and trench depths measured from vallatescross-sectioned at a thickness of 20 or 30 mm, and stained with 1:60MAb Troma-I or with antiserum against PGP9.5 at concentrations of1:800, 1:1200, or 1:1600.

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–96 89

mouse was determined after the behavioral tests had beencompleted.

3. Results

3.1. A brief surÕey of Õallate morphology in bdnf wild-typemice

The dorsal surface of the posterior portion of the mousetongue contains a solitary vallate whose papilla is brack-

eted by two rostro-caudally directed trenches. The innerand outer walls of the right and left trenches are thevallate’s principal gustatory epithelium. The trench wallscontain more than 90% of the vallate taste buds. Theremaining vallate taste buds are located on the dorsal

Ž .surface of the vallate papilla. At birth P0 the trench wallshave substantial innervation and an average of about 6taste buds. The trenches open shortly after birth. By P7 awild-type mouse has a wide vallate papilla bracketed bydeep trenches whose heavily innervated walls contain morethan 100 taste buds.

Fig. 5. Cross-sections of mouse vallate papilla are stained with polyclonal antiserum against PGP9.5 at a dilution of 1:1600. The total number of vallatetaste buds counted in complete serial sections is indicated in the lower right of most panels; panels with identical numbers represent sections from the same

Ž . Ž . Ž . Ž .vallate. Innervation patterns are shown for the entire vallate A–D , a portion of the trench wall E–G , and a representative taste bud H–K . Aqrq Ž . yryExtensive innervation of a P7 bdnf vallate. Four taste buds are indicated with arrowheads. B A well-innervated P7 bdnf vallate with two tasteŽ . yrybuds indicated with arrowheads. C Arrows indicate two supernumerary trenches in a P7 bdnf vallate papilla, with their epithelial downgrowths

Ž . yryindicated by smaller arrows below. Sparse innervation of a taste bud is indicated by an arrowhead. D This section of P7 bdnf vallate has no axons inŽ .the left trench open arrow . The two pairs of solid arrows indicate fringing epithelial innervation that, in tandem with about half of the dorsal vallate’s

Ž . Ž . qrqinnervation arrowhead , disappears completely in nt3 null mutants; see Section 3.4. E Innervation of a portion of the vallate trench wall in a P1 bdnfŽ .mouse. The palisade array of axons solid arrows is a common prelude to taste bud formation. A taste bud contains larger axons with multiple varicosities

Ž . Ž . Ž . Ž . yry Ž .open arrows . F Small caliber axons solid arrows innervating the trench wall and a nascent taste bud open arrow in a P7 bdnf mouse. G Theyry Ž .trench wall in a P7 bdnf mouse was innervated either by two small caliber axons solid arrows or perhaps by three axons that separated above the

Ž . qrq Ž . Ždouble headed arrow. H In a P7 bdnf vallate several varicosities are evident in a small axon smaller pair of arrows and a large axon larger pair of. Ž . qrq Ž . yryarrows . I A vallate taste bud of a P7 bdnf mouse is replete with axons and their varicosities. J, K In an individual taste bud of P7 bdnf vallate

Ž . yryopen arrows , the density of varicosities was diminished as were the numbers of vallate taste cells. Axons entering a bdnf taste bud are indicated by aŽ . Ž . Ž .pair of solid arrows in K . Scale bar in G is 90 mm for A–D and 20 mm for E–K .

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–9690

3.2. Bdnf yry Õallate papilla morphology

In comparison with wild-type mice, P7 bdnfyry micehad a narrow vallate papilla, with shallow trenches, sparseinnervation, and markedly fewer taste buds. These features

Žwere evident in tongue sections stained for taste buds Fig.. Ž .1 and in sections stained for axons Fig. 5A–D . In null

Žmutants, the residual intragemmal cells cells within the. Ž .taste bud had the characteristic elongated shape Fig. 1C

Ž .and were gustducin-immunoreactive data not shown .In most bdnfyry mice, the vallate papilla was stunted

in width and height, sometimes remaining below the levelof the tongue surface. As a common accompaniment ofsuch stunted vallate papillae, the vallate’s dorsal surfacewas creased by one or two shallow supernumerary trenchesthat generally ran parallel to the normal trenches. Theepithelial morphology of these supernumerary trenchessuggested that, like normal trenches, they were epithelial

Ž .downgrowths Fig. 5C . When viewed through a dissectionmicroscope, a bdnfyry mouse tongue having a narrowvallate papilla that bore a supernumerary trench was apredictor of the following additional defects: shallow rightand left trenches with a reduced trench wall area, a signifi-cant shortfall of taste buds, and sparse innervation. Toquantify bdnfyry vallate structural defects, morphometric

Ž .measurements were made at different ages Table 1 . Thepresence of lingual abnormalities at birth indicates that thevallate papilla’s structural defects arose during gestation,several days before the formation of most vallate tastebuds.

Table 1Vallate structure and taste bud abundance in bdnfyry and bdnfqrq micemeans"standard deviations are shown

a aP0 P7 P12

( )Papilla width mmŽ . Ž . Ž .yry 129"32 ns3 185"47 ns9 220"10 ns5Ž . Ž . Ž .qrq 179"8 ns3 260"34 ns10 330"43 ns5

Percent 72% 71% 65%

( )Trench depth mmŽ . Ž .yry y 95"34 ns9 80"15 ns5Ž . Ž .qrq y 145"21 ns9 182"50 ns5

Percent – 66% 44%

2( )Trench wall area 1000 mmŽ . Ž .yry y 13.7"9.0 ns16 12.1"5.1 ns10Ž . Ž .qrq y 21.3"6.6 ns8 34.7"7.2 ns8

Percent – 64% 35%

bNumber of taste budsŽ . Ž . Ž .yry 1"1 ns3 24"22 ns11 12"10 ns5Ž . Ž . Ž .qrq 6"2 ns3 101"14 ns7 123"14 ns5

Percent 17% 24% 10%

a Differences between bdnfyry and bdnfqrq mice in papilla width,trench depth, trench wall area, and number of taste buds are significant atP -0.01, except P -0.05 for trench wall area at P7.b Trench wall taste buds only; the 0–10 dorsal taste buds are not included.For trench wall area, nsnumber of trenches.

The bdnfyry newborn’s sparse innervation, shortfall ofvallate taste buds and reduced gustatory epithelial area didnot recover postnatally, e.g., between P7 and P12. Al-though bdnfyry mice grew larger from P7 to P12, theirvallate’s dimensions continued to decline as a percentageof the wild-type. For example, compared with wild-type,the trench wall epithelial area was reduced by 35% at P7and by 65% at P12, in spite of a 20% increase from P7 toP12 in the absolute width of the null mutant tongue at the

Ž .level of the vallate ns3 mice . The small size of thebdnfyry mice does not fully explain the stunting of thevallate. For instance, compared with P7qrq mice, theP7yry vallate papilla was 30% narrower, whereas theentire tongue was only 10% narrower at the level of thevallate. Moreover, the slower growth of bdnfyry micecannot account for any defect like a supernumerary trenchthat was wholly abnormal.

3.3. Bdnf yry Õallate taste buds

Since wild-type controls had few taste buds in thevallate trenches at birth, the relative abundance of tastebuds at P7 and P12 was a more useful test of the shortfallof taste buds in bdnfyry mice. Shortfalls were oftengreater than 90% at P7; it was not unusual for a bdnfyry

trench to contain 0–4 taste buds rather than the normal50q . However, some P7 null mutants had ample vallate

Žinnervation and more than 25 taste buds per trench theimplication that taste neurons and taste buds were some-

.times rescued is considered in Section 4 . On average, nullmutant vallate taste buds were not only less abundantŽ . Ž .Table 1 , they were also smaller Fig. 2 , and depleted ofintragemmal cells.

To determine whether smaller trench walls were limit-ing the number of taste buds, we made histological recon-structions of the trench wall. Though small in area, thebdnfyry trench walls would have been physically able toaccommodate additional taste buds beyond the scattered

Ž .few that were present Fig. 3 .yry ŽFor 12 bdnf mice in Fig. 4, the best fit line heavy

.dashes indicates that the number of taste buds was almostŽ .in exact proportion dotted line to trench wall area. The

parallel slopes of the short lines connecting the data pointsfor the right and left trench of individual mice demonstrate

Žproportionality within each null mutant mouse for 9 of 12.mice . The minimal area for a trench wall to support one

2 Ž .taste bud was 2100 mm X intercept . We next sought toevaluate whether innervation density was the commoncontrolling factor responsible for the covariance of tastebud abundance and trench wall area.

3.4. InnerÕation-dependence of Õallate defects

The vallate trench walls of P7 bdnfqrq mice hadnumerous taste buds and axons. In P7 bdnfyry mice, a

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–96 91

smaller vallate papilla and fewer taste buds were bothassociated with diminished innervation whether examined

Ž .within the entire vallate left column, Fig. 5 , within theŽ .trench walls middle column, Fig. 5 , or within taste buds

Ž .right column, Fig. 5 . Given the scarcity of axons in theyry Ž .bdnf trench wall Fig. 5E–G , it was not surprising

that there were correspondingly fewer varicosities in trenchŽ .wall taste buds Fig. 5I–K . In the most severe pheno-

types, only a few axons entered the residual taste budsŽ .Fig. 5K .

While it was possible to rank trench wall axon densityŽ .as high, medium, or low e.g., Fig. 5F–G , it was not

possible to count accurately the number of trench wallaxons since they were often twisted into bundles thatcoursed into adjacent sections. To obtain a more reliablemeasure of gustatory innervation density, per se, wecounted the axon varicosities clustered within taste buds inthe gustatory epithelium. A varicosity was defined as aglobular axonal swelling extending at least 1 mm in alldirections. We restricted our quantitative correlations tothe trench walls where there were few somatosensoryaxons that might confound appraisal of the correlationbetween gustatory innervation density and morphology.

The number of taste buds increased as a linear functionof taste bud innervation density in P7 bdnfyry miceŽ .rsq0.96, Fig. 6A . Thus, taste bud varicosity densitywas a useful proxy for vallate trench wall innervation. Inwild-type mice, varicosity density appeared to saturate atthe higher vallate innervation levels, since P7 bdnfqrq

mice averaged more than 100 trench wall taste buds, buthad as few as 35 intragemmal varicosities per 1000 mm2.Nonetheless, when the trench wall innervation was sparse

Ž .to moderate i.e., not abundant , the number of intragem-mal varicosities per unit area served as a quantifiablegauge of innervation density of the gustatory epithelium.

The area of the gustatory epithelium also increased as aŽlinear function of taste bud innervation density rsq0.94,

.Fig. 6B .Putative somatosensory axons persisted on the dorsal

Ž .surface of the vallate papilla arrowhead in Fig. 5D andŽon the dorsal surface of the outer trench walls vallate

. yryfringing epithelium, solid arrows in Fig. 5D . In nt3mice, innervation of the fringing epithelium was totallymissing, along with about half of the innervation to thedorsal surface of the vallate papilla proper, whereas the

w xgustatory innervation seemed unimpaired 63 . Since thevallate papilla’s dorsal surface probably included a mixture

Ž .of NT3-dependent somatosensory axons and BDNF-de-pendent taste axons, the depleted gustatory innervation inbdnfyry mice was more evident in the paucity of axons

Ž .within the papilla’s core and trench walls e.g., Fig. 5 .

3.5. ObserÕations on other gustatory papillae

Although this report places its emphasis upon the val-late papilla to complement previous postnatal studies, weobserved that gustatory development was also impaired atother locations. The bdnfyry foliate papillae had threedefects characteristic of the vallate papilla. Specifically,the bdnfyry foliate papillae had reduced innervation, fewertaste buds and shallower trenches. The nasopalatine papilla

whad fewer taste buds than normal e.g., at P7: 12"7Ž . Ž qrq .xmean"1 S.D., ns5 vs. 33"11 bdnf , ns5 . Arange of deformity was evident in fungiform taste papillae.

Ž . 2 Ž .Fig. 6. A The number of varicosities per 1000 mm of taste bud area in the largest section was linearly related to the total number of vallate taste budsŽ yry . Žns5 P7 bdnf mice . Each data point represents, for one mouse, the total number of its vallate trench wall taste buds and innervation density the

.mean"1 S.E. of the varicosities per unit area of taste bud profile . Varicosities were counted in about 50% of the trench wall taste buds; sections of theremaining taste buds had indistinct boundaries or had sectioning defects. The straight line is the best fit line, rsq0.96. The data were obtained from five

yry Ž .uniformly processed P7 bdnf vallate papillae sectioned at 20 mm and stained with antiserum against PGP9.5 at 1:1600 dilution. B Innervationyry Ž . Ž .density for the same five P7 bdnf mice as in A was linearly related to the combined areas of the right and left trench walls rsq0.94 .

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–9692

Some fungiform papillae were entirely missing, especiallythose more than 3 mm caudal to the tip of the bdnfyry

tongue. Such caudal fungiform papillae seem to havedeveloped into filiform papillae. The conversion of fungi-form to filiform papillae is a standard outcome of the

w xpostnatal denervation of rat papillae 21,22,35,36,45 . Rel-atively normal bdnfyry fungiform papillae and taste budswere also observed. Similar results have been obtained for

w xfungiform papillae in trkB null mutants 20 . The smallestbdnfyry fungiform taste ‘buds’ consisted of only 1–2keratin 8-immunoreactive receptor cells. While residualfungiform papilla were smaller in some of the bdnfyry

mice, interpretation is confounded by the concurrent obser-vation that about 25% of wild-type mice also had smallfungiform papillae—possibly a recessive trait independentof BDNF.

3.6. BehaÕioral tests of neonates

The modern confirmations that urodele taste buds candevelop without innervation raised expectations that mam-

w xmalian taste buds may develop without innervation 4–6 .Given the paucity of axons in the gustatory epithelium ofbdnfyry mice, it was of interest to determine whetherresidual bdnfyry taste buds were functionally innervated.The selective ingestion of a drop of sucrose was anefficient and direct test of functional gustatory innervation.If bdnfyry mice behaved toward sucrose like normal micethat uniformly prefer its sweet taste, it would unequivo-cally indicate that there were functional neural connectionsboth to taste buds and to the brain. Taste behavior wasevaluated in 13 litters comprising 101 P7-P14 neonates.Gentle handling and minimal physical restraint were essen-tial for obtaining fluid intake. Even so, about half of the101 mice did not respond to water or to sucrose. Thesemice may have been sated with milk. The wide range ofunresponsiveness, from a low of 10–12% in four litters toa high of 80% in two litters, is consistent with variation insatiety. A higher percentage of bdnfqrq than bdnfqry orbdnfyry mice failed to respond to the solutions; perhapsbecause the bdnfqrq mice were more effective at nursing,

Table 2Categorization of behavioral responses to water and 0.3 M sucrose forwild-type, heterozygous, and homozygous BDNF null mutant neonates

Ž . Ž . Ž .aged P7 ns16 , P12 ns53 , or P14 ns32

Genotype Total

qrq qry yry

Number of mice

39 42 20 101

Response categoriesSelective intake of sucrose 30% 41% 30% 33%No intake of water or sucrose 57% 42% 40% 49%Intake of water and sucrose, 13% 17% 30% 18%or of water only

Table 3Taste bud abundance and selective sucrose intake in a litter of P12 BDNFmice

aAnimalrgenotype Number of taste buds Water 0.3 M sucrose

Val Fun NP

B337 yry 9 69 4 0 qqB336 yry 29 58 10 0 qqB333 qrq 146 141 42 0 qB332 qrq 123 )100 41 0 qB330 qrq 0 qqB335 qrq 0 qqB329 qry 0 qqB331 qry q qqB334 qry 0 0

a Taste bud counts in the vallate, fungiform, and nasopalatine papillae arebilateral.0sno response, qsbrief, moderate ingestion, qqsprolonged, vig-orous ingestion.

and therefore more likely to be sated at the time of testing.Contrasting completely with an absence of drinking, wasthe indiscriminate drinking anticipated in instances wheremilk deprivation produced thirst sufficient to prompt wateringestion: about one fifth of the neonates ingested bothwater and sucrose, or ingested only the initial water solu-tion. It was the selective responsiveness of the remainingthird of the mice that was informative. Specifically, at least30% of each genotype selectively ingested the two sucrosepresentations from among the six taste solutions sequen-tially offered. Independent observers judged the latencyand duration of suckling movements to the small drop ofsucrose to be similar among the three genotypes. Thecategorization of responses by genotype is shown in Table2.

To demonstrate taste bud shortfalls in specific bdnfyry

mice that drank sucrose but not water, we counted tastebuds in two bdnfyry and two bdnfqrq mice from one P12

Ž .litter Table 3 . In this litter, 7 of the 8 mice that readilyingested sucrose ignored water. One bdnfyry mouse,which selectively ingested sucrose, had only a few na-sopalate or vallate taste buds, but numerous fungiformtaste buds. In this and other litters, the vigorous ingestiveresponses to sucrose were comparable in bdnfyry,

qry qrq w xbdnf , and bdnf mice. Nosrat et al. 37 were unsuc-cessful in obtaining selective responses to taste solutionspipetted into the mouth of restrained P12–P19 bdnfyry

neonates, perhaps as a result of restraint-induced distressand the greater vestibular disorientation of older neonates.

4. Discussion

The present findings on the mouse gustatory systemŽ .exemplify three developmental principles: i subtypes of

sensory neurons are dependent upon specific neu-Ž .rotrophins; ii sensory innervation contributes to the mor-

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–96 93

Ž .phogenesis of skin; and also iii contributes to the mor-phogenesis of sense organs.

Ž .i Vallate taste neurons are dependent on BDNF. Sometaste neurons were rescued. The vallate receives innerva-tion from a mixed population of IXth nerve sensory axons.About half of the normal innervation of the dorsal surfaceof the vallate papilla proper, and all of the innervation of

Žthe dorsal fringing epithelium across the trench from the.papilla was NT3-dependent since it disappeared from

yry yry w xnt3 mice but not from bdnf mice 63 . The com-bined absence of taste buds and axons in the trench wall ofsome bdnfyry mice suggests that trench wall innervationis largely restricted to taste axons that require BDNF.However, at least a few intragemmal axons and occasionaltaste buds survived in most, but not all, bdnfyry trenchwalls. The abundance of residual taste buds was highlycorrelated with innervation density. It is unlikely that theseresidual taste axons were a BDNF-independent subset ofgustatory neurons, given that some bdnfyry vallatetrenches were devoid of axons. It is more likely thatvariable numbers of BDNF-dependent gustatory neuronschanced to be rescued by NT3 or NT4 that also bind to

w xaxonal trkB receptors 28 .Ž .ii BDNF-dependent gustatory innervation contributes

to the morphogenesis of gustatory epithelia. Neurotrophinsare characteristically viewed as survival factors that selec-tively sustain subsets of developing neurons or their pre-

w xcursors 10 . Consequently, most studies of neurotrophinnull mutant mice have focused upon defects in neuronsrather than in other tissues. However, the nerve-depend-ency of tissue development is a quietly persistent theme indevelopmental biology that prompts quests for defects innon-neural tissues. Examples include the lethal nt3yry

heart malformations that may stem from deficient cardiacw xneural crest cells 12 . Cutaneous overexpression of NT3

increases both the abundance of cutaneous innervation andw xthe size of touch domes 3 . The arrival of glabrous skin

sensory axons into fetal monkey digits is associated withepidermal cell proliferation and repositioning of papillary

w xsweat duct ridges 11 .The present investigation has revealed that the normal

morphogenesis of lingual gustatory epithelia, as a type ofŽw x w x.moderately keratinized skin 30,62 , cf. 61 requires

BDNF-dependent taste neurons for robust cutaneous devel-opment. While BDNF might, in principle have directeffects on the vallate trench wall, this would not accountfor the variable severity in the papilla’s defects althoughBDNF was always absent. Instead, the positive correlationbetween defect severity and depleted innervation suggeststhat the papilla’s defects resulted from sparse innervation.It is possible that papilla positional specification is alreadyunderway prior to nerve fiber arrival, since the mousevallate epithelium has already thickened, and putative sitesof mouse fungiform papillae express sonic hedgehog and

w xbone morphogenetic protein-4 mRNA 1,8,48,53 . Moretellingly, tongue fragments removed before innervation

and subsequently organ cultured in the absence of innerva-w xtion give rise to nascent fungiform papillae in vitro 17,33 .

Even if the placement of gustatory papillae and their initialŽvisible emergence reflect autonomous epithelial events or

.mesenchymal–epithelial interactions , it is still necessarythat subsequent axonal–epithelial interactions sustain gus-tatory papilla development and prevent: the formation ofsupernumerary trenches, the losses of fungiform papillae,and the reductions in vallate papilla width and trench wallarea that typify the bdnfyry phenotype.

Periods of early postnatal denervation can also deformw xgrowing vallate papillae 42 . Similarly and better docu-

mented, the postnatal development and maintenance offungiform papillae and taste buds are nerve-dependent to adegree that varies with species and diminishes with agew x7,21,22,35,36,45,47,52,53 . In P1 rats, chorda-lingualnerve transection eradicated all fungiform taste buds andtransformed all fungiform papillae into filiform papillae.As neonatal rats matured, denervation was progressively

w xless effective in eliminating fungiform taste buds 35 . The100% nerve-dependence of P1 fungiform taste buds con-flicts with postulations of late gestational nerve-indepen-

w xdence 6,20 .Gustatory, rather than somatosensory, axons sustain the

developing vallate papilla, since the absence of NT3-de-pendent somatosensory axons in nt3yry mice did notnoticeably impair vallate development examined at P0–P4Ž .unpublished observations . Nor did the presence of NT3-sustained somatosensory axons prevent the stunting of thedeveloping bdnfyry vallate papillae. Similarly, so-matosensory axons, such as those of the lingual or auricu-lotemporal nerves, were less than 2% as effective asgustatory axons in sustaining fungiform papillae by sup-pressing ectopic filiform spine outgrowth from denervated

w xfungiform papillae of adult rats 45 .Ž .iii Taste bud development is nerve-dependent. It has

been widely appreciated that the morphogenesis of varioussecondary receptor cells is nerve-dependent in mammalsw x57 . The general importance of innervation for the fetaldevelopment of cutaneous secondary sensory cells hasreceived recent support from the study of trkC and NT3null mutant mice. In both instances, proprioceptive neu-rons die and muscle spindle and golgi tendon organ targets

w xfail to develop 15,18,29,31,57 . Presumably, peripheraltarget tissue delivers NT3 to the proprioceptive neuronsthat in turn deliver unknown nerve-derived trophic factorsthat promote muscle spindle and tendon organ maturation.

The abundance of vallate taste buds in bdnf null mutantmice was positively correlated with innervation density.Those bdnfyry mice with only a few vallate taste budshad only a few axons in the gustatory epithelium, and onlya few varicosities in their taste buds. The sparse innerva-tion within vallate taste buds of bdnfyry mice was alsorevealed by double staining for taste receptor cells and

w xintragemmal axonal neurofilaments 63 . Taste axons musthave carried appropriate sensory messages to the brain,

( )B. Oakley et al.rDeÕelopmental Brain Research 105 1998 85–9694

since bdnfyry mice selectively and promptly ingestedsucrose in a simple behavioral test. Functionally innervatedfungiform taste buds were the most likely mediators of thisprompt sucrose ingestion. It would have taken severalmore seconds for sucrose to reach the vallate or foliate

Žw xtaste buds, and produce a drinking response 44 and.unpublished electrophysiological latencies . Situated ante-

riorly, the nasopalatine papilla would also be promptlyexposed to taste solutions, but its taste buds were substan-tially depleted in some sucrose-responsive bdnfyry mice.

Since there was already a significant shortfall of tastebuds by birth, the depletion of taste neurons must alsoimpair prenatal taste bud development. Prenatal nerve-de-pendence of taste bud development is fully concordantwith the present results and with earlier demonstrations ofpostnatal nerve-dependence where denervation both elimi-nated existing fungiform and vallate taste buds and denied

w xtheir further development 35,41 . Further, after a P0–P10period of experimental denervation, the vallate gustatoryepithelium permanently lost its competence to support

w xtaste buds 42 .

4.1. The temporal sequence of eÕents in Õallate gustatorydeÕelopment

We outline six of the probable sequential steps innormal mouse vallate gustatory development, as compiled

w xfrom several studies 1,8,23,38,51,54 and our unpublishedŽ .observations: step 1 vallate gustatory neurons proliferate

in the petrosal ganglion as future IXth nerve components;Ž .2 the site at which the vallate gustatory papilla will

Ž .develop is specified; 3 axons reach the deeper mesenchy-Ž .mal tissue around E11-12; 4 vallate-derived BDNF sus-

Ž .tains taste neurons after E13; 5 The vallate trench arisesŽ .from an epithelial downgrowth that begins at E14; 6 the

first keratin 8-positive taste cells appear just before birth,at least 5 days after the trench is first evident. By birth,most wild-type mice have several taste buds in the vallate

Žw x .trench walls 63 ; Table 1 and unpublished observations .

4.2. A proposed model of Õallate deÕelopment

The empirical nerve-dependency of prenatal and postna-tal gustatory morphogenesis prompts a model consistentwith the suggested temporal progression of normal devel-opmental events outlined above. The model in Fig. 7begins with BDNF since there is little or no informationabout the roles of other pre-innervation lingual factors thatmake early contributions to the cell lineages, or to othertransformations that foreshadow vallate papilla develop-ment. The model allows both for pre-innervation signalingmolecules in the tongue that specify the vallate’s site andrudimentary form, and also for unknown rescue factorsŽ .perhaps NT3 that salvage some BDNF-dependent tasteneurons. The central tenets of this model are that BDNF-

Fig. 7. Schematic depiction of causal steps in the postulated partialdependence of vallate morphology and taste bud formation upon BDNF-dependent taste neurons. The Pearson correlations relating to innervationdensity are derived from the five uniformly assessed animals in Fig. 6.For the regression of trench wall area on the number of taste buds pertrench, r of q0.71 comes from the 12 mice in Fig. 4, rather than the five

yry Ž .P7 bdnf mice in Fig. 6 r sq0.83 .

dependent taste neurons are necessary both for completingthe development of this specialized epithelial papilla andfor the development of its taste buds.

As previously indicated, developmental nerve-depend-encies of skin and secondary sense cells are not unique tothe peripheral gustatory apparatus. Moreover, the out-

Žcomes of experiments on amphibians, both classical re-w x.viewed in Ref. 43 and modern, are concordant in reveal-

ing that neither the development nor the maintenance ofw xurodele taste buds is nerve-dependent 4–6,49,55 . Among

mammals, by contrast, the outcomes of classical experi-Ž w x.ments reviewed in Ref. 40 and modern experiments

Žwith postnatal and prenatal denervations reviewed in Ref.w x.41 , with nerve-free organ cultures, and with in vivoassessment of bdnf null mutant mice are consistent withthe causal sequence in which BDNF sustains taste neuronsthat in turn contribute to gustatory papilla developmentand subsequently to taste bud development.

Acknowledgements

Supported in part by grants from the National ScienceŽ .Foundation bir-9413211 and the National Institute on

Ž .Deafness and other Communication Disorders DC00083 .

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