ORIGINAL PAPER

Donald Ganchrow Æ Judith Ganchrow

Martin Witt Æ Eve Arki-Burstyn

The effect of b-bungarotoxin, or geniculate ganglion lesion on taste buddevelopment in the chick embryo

Accepted: 10 March 2006 / Published online: 31 May 2006� Springer-Verlag 2006

Abstract Chick taste bud (gemmal) primordia normallyappear on embryonic day (E) 16 and incipient immature,spherical-shaped buds at E17. In ovo injection of b-bungarotoxin at E12 resulted in a complete absence oftaste buds in lower beak and palatal epithelium atdevelopmental ages E17 and E21. However, putativegemmal primordia (solitary clear cells; small, cellgroupings) remained, lying adjacent to salivary glandduct openings as seen in normal chick gemmal devel-opment. Oral epithelium was immunonegative to neuralcell adhesion molecule (NCAM) suggesting gemmalprimordia are nerve-independent. Some NCAM immu-noreactivity was evident in autonomic ganglion-like cellsand nerve fibers in connective tissue. After unilateralgeniculate ganglion/otocyst excision on E2.5, at devel-opmental ages E18 and posthatching day 1, �12% ofsurviving ipsilateral geniculate ganglion cells sustained�54% of the unoperated gemmal counts. After E18,proportional stages of differentiation in survivingdeveloping buds probably reflect their degree of inner-vation, as well as rate of differentiation. Irrespective ofthe degree of geniculate ganglion damage, the propor-tion of surviving buds can be sustained at the samedifferentiated bud stage as on the unoperated side, ormay differentiate to a later bud stage, consistent with the

thesis that bud maturation, maintenance, and survivalare nerve-dependent.

Keywords Taste bud Æ Chick Æ Geniculate ganglion Æb-Bungarotoxin Æ NCAM Æ Vimentin

Introduction

The role of gustatory nerves in initiating taste budmorphogenesis has long been implied, in part because ofthe virtual coincidence in oral epithelium of the tastebud (gemmal) primordium with the invasion of tasteafferents into epithelium (Farbman 1965; Farbman andMbiene 1991; Whitehead and Kachele 1994; Witt andReutter 1996, 1998; Witt and Kasper 1999; El-Sharabyet al. 2004; Northcutt 2005; cf. Torrey 1940; Ramon yCajal 1929). However, reports in the amphibian sala-mander (Stone 1933, 1940; Barlow et al. 1996; Barlowand Northcutt 1997; Northcutt and Barlow 1998), pis-cian channel catfish (Northcutt 2004) and mammalianrodent (Fritzsch et al. 1997) have suggested that tastebud progenitor cells appear independent of innervation,presumably forming within local oral ectoderm, oro-pharyngeal endoderm (Stone et al. 1995; Barlow andNorthcutt 1997), or trunk ectoderm (Northcutt 2004). Inmouse embryonic tongue epithelium, more than one-third of cytokeratin 8-positive, presumably identifiedgemmal cells appear prior to neural invasion of the oralepithelium (Mbiene and Roberts 2003). The issue ofinitial taste bud formation should be distinguished froma neural maintenance function apparently essential forachieving gemmal maturity, and survival once the tastebud primordia form (e.g., Ganchrow et al. 1986;Ganchrow and Ganchrow 1987). For example, it hasbeen shown that the first 3 postnatal days constitute acritical period in which unilateral glossopharyngealnerve afferentation assures the subsequent adult com-plement of buds in rat circumvallate papillae (Hosleyet al. 1987). Further, disruption of neurotrophin genes inBDNF- or TrkB receptor-null mutant mice results in

D. Ganchrow (&)Department of Anatomy and Anthropology,Sackler Faculty of Medicine, Tel Aviv University,69978 Ramat Aviv, Tel-Aviv, IsraelE-mail: dganchrow@ucsd.eduTel.: +1-858-5345943Fax: +1-858-5345943

J. Ganchrow Æ E. Arki-BurstynInstitute of Dental Sciences,The Hebrew University-Hadassah School of DentalMedicine Founded by the Alpha Omega Fraternity,P.O.B. 12272, 91120 Jerusalem, Israel

M. WittSmell and Taste Clinic, Department of Otorhinolaryngology,and Department of Anatomy, University of DresdenMedical School, Fetscherstrasse 74, 01307 Dresden, Germany

Histochem Cell Biol (2006) 126: 419–435DOI 10.1007/s00418-006-0177-2

reduction in the number of fungiform and circumvallatetaste bud-bearing papillae and degree of their intra-gemmal innervation (Fritzsch et al. 1997; Nosrat et al.1997; Mistretta et al. 1999; reviewed in Oakley and Witt2005).

One way to investigate the role of nerve in initiatingtaste bud formation is to arrest the potential (chemo-sensory) nerve–epithelial interactions in advance of theappearance of the taste bud primordium. In embryonicrat, it has been shown that the injection of snake venom-derived neurotoxin (Chang and Lee 1963), b-bungaro-toxin (b-BT; 0.4, or 1.0 lg/ll, i.p.), between embryonicday (E) 13 and E17 and animal utilization 4–7 dayspostinjection, destroys somatic nerves as well as somatain sensory and autonomic ganglia, blocks muscle spindleformation, and destroys spinal cord somatomotor cells(Hirokawa 1978; Harris 1981; Tummers et al. 1986;McCaig et al. 1987; Condon et al. 1990; Kucera andWalro 1990a, b; Ciutat et al. 1996). Similarly, inembryonic chick, cumulative b-BT injections (0.40 lg/ll, into the yolk sac) administered every 3 days betweenE4 and E17 yielded total destruction of all cranial nervesincluding acoustic and vestibular ganglia and theirproximal nerve terminals. Interestingly, however,peripheral sensory auditory and vestibular hair cells aswell as olfactory epithelium receptor neurons wererefractory to the neurotoxic effects of b-BT (Hirokawa1977, 1978). The sole report on the effect of b-BT in thedeveloping peripheral taste system indicated that miceinjected with b-BT (1.0 lg/ll, into the amniotic cavity)on E12 showed a complete loss of sensory (lingual andglossopharyngeal nerves) and motor (hypoglossal nerve)nerves in the tongue including complete loss of normallyidentifiable, anterior tongue fungiform papillae, poster-ior tongue circumvallate papillae and palatal taste budsof ‘adult’ morphology at E18, 1 day prior to birth(Morris-Wiman et al. 1999). Intriguingly, signs wereevident of fungiform and palatal taste bud-like primor-dia (Morris-Wiman et al. 1999).

The embryonic chick is an important vertebratemodel to test whether taste bud primordium formationis nerve- or papilla-independent (cf. Farbman andMbiene 1991; Mbiene and Roberts 2003) since the lowerbeak as well as palatal buds do not reside within bud-bearing intraoral papillae. As in the rat, the chick ges-tation (incubation) period is 21 days, but unlike thealtricial rat (Mistretta 1972; Hosley and Oakley 1987)precocial hatchlings already exhibit the adult comple-ment of taste buds (Ganchrow and Ganchrow 1987).Chick taste bud primordia are initially evident in oralepithelium on the 16th embryonic (E16) day of incuba-tion as a small grouping of cells deep in surface epithe-lium, and characteristically located adjacent to salivarygland duct openings onto surface epithelium (e.g., Saito1966; Berkhoudt 1985; Ganchrow and Ganchrow 1985,1987). By E17, a more clearly defined spherical cluster ofbud cells containing a tubular lumen is seen deep to thesuperficialmost layer in oral epithelium. Taste buddevelopment is rapid during the last 4 days in ovo

(Ganchrow and Ganchrow 1987), such that at E19 thebud attains a more ovoidal shape that may extend theentire vertical depth of oral epithelium, with the gemmallumen continuous at surface epithelium at the taste pore.By E21, most taste buds have attained the matureovoidal shape (see Materials and methods), with gemmaldiameter stabilizing by the fourth posthatching day(Ganchrow et al. 1994). With respect to developinginnervation of chick taste buds in oral epithelium, earlystudies suggested that already at 72 h of incubation (endof E3), incipient distal and proximal processes of thegeniculate ganglion anlage may be traced to the firstectodermal (epibranchial) placode and third rhombo-mere, respectively (Tello 1923). Later immunocyto-chemical studies using anti-neurofilament proteinantibodies showed that at E4, a distinct chorda tympaninerve branch is seen arising from two looping distalprocesses of the geniculate ganglion in the region of theotic vesicle (otocyst), and by E5, the nerve has reachedthe pharyngeal floor of the developing mandible(Kuratani et al. 1988; Kuratani and Tanaka 1990). Inturn, the chorda tympani presumably courses within thepostero-anterior trajectory of the developing mandiblethrough at least E8 [when the leading edge of the man-dible finally contacts the upper beak (Hamburger andHamilton 1951)], eventually to innervate taste buds inthe epithelium of the anterior floor of the lower beak(Gentle 1983; Gentle and Hunter 1983; Ganchrow et al.1986). Previous ultrastructural analyses indicate thatnerve profiles are evident in chick embryonic taste budsby E18 (Ganchrow and Ganchrow 1987). Proximally, byE4, the geniculate ganglion proper connects to therhombencephalon by a long myelinated nerve root (vanCampenhout 1937) and the solitary tract in caudalbrainstem appears complete by E9 (Bok 1915).

Source cells of chick geniculate ganglion neurons arederived from the pharyngeal endoderm-induced (Begbieet al. 1999), first epibranchial placode, which liesimmediately anteroinferior to the otocyst (Yntema 1937,1944; D’Amico-Martel and Noden 1983; cf. Northcutt2004). All geniculate ganglion somata are postmitotic byE5 (D’amico-Martel 1982). Though the time at whichgustatory nerve invasion of lower beak oral epitheliumhas not yet been established for the chick, in the rat withthe same gestation period of 21 days as in the chick, thechorda tympani is first evident in lingual epithelium byE17–18 (Farbman 1965; Farbman and Mbiene 1991);[cf. mouse, 19 days gestation, at E14.5 (Mbiene andRoberts 2003); hamster, 15–16 days gestation, at E13(Whitehead and Kachele 1994)].

Thus, based mainly on the aforementioned studies inchick and rodent embryos, in the present study, it waspresumed that a single injection in ovo of b-BT in chickembryos at E12/E13, prior to incipient taste bud devel-opment at E17 (Ganchrow and Ganchrow 1987), woulddestroy, or at least arrest further distal development ofthe chorda tympani and greater superficial petrosalnerves and their source somata in the geniculate gan-glion prior to their respective neural penetration into

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mandibular and palatal oral epithelium. If taste budprogenitor cells reside in local epithelium, it could behypothesized that in the absence of gustatory innerva-tion, gemmal primordia might still be evident. Further,in a separate set of experiments, it was reasoned thatgeniculate ganglion destruction via otocyst removal atE2.5 would eliminate most early-growing innervation toprospective early-forming gemmal primordia. Unpub-lished data (Ganchrow and Ganchrow) suggested thatafter such early surgical lesion of the geniculate gan-glion, the earliest (spherical) stage of taste bud devel-opment at E17–E18 might still appear. If the gustatorynerve is essential for taste bud survival, it could behypothesized that absence of, or insufficient geniculateganglion innervation might arrest further gemmal dif-ferentiation and result in subsequent taste bud degen-eration by E21. The present study thereby describes andcompares the effects of neurotoxin-initiated denervationand surgical destruction of the geniculate ganglion ongeniculate ganglion and taste bud development in lategestation embryos.

Materials and methods

Animals

Fertilized eggs of the White Leghorn, Anak (broiler)strain, obtained from the Kibbutz Tzuba hatchery(Jerusalem), were placed in a forced-draft portableincubator (Victoria, Pavia, Italy) at 37.6�C and 84%relative humidity. Individual eggs were rotated every15 min and the resting position of the two-sided base ofthe incubator changed every hour, daily during daylighthours, until either b-BT injection at E12, or otocyst le-sion at E2.5 (see below). The first 24 h of incubationwere considered as E1. In order to compare these results

with mammalian embryonic data, studies cited whichreported an E¢0¢, i.e., the first 24 h after the night ofmating, have been redefined as E¢1¢.

b-BT injection

On chronologic (gestation) day E12 (see Morris-Wimanet al. 1999 in mouse whose gestation period is about19 days), eggs were removed singly. After localizedswabbing with 70% alcohol and the blunt side of theeggshell pierced so as to permit greater stabilization ofthe embryo, the shell was windowed (�3–4 mm2) with a#10 scalpel blade, and the underlying outer shellmembrane incised thereby exposing the vascularizedchorioallantoic membrane. Under the visual guidanceof a Zeiss operation microscope, either 1 ll of 0.9%sterile sodium chloride (B-Braun Melsungen AGD34209) in five sham-operated control, or 1 lg of b-BT(Sigma, catalogue number T5644) dissolved in 1 llsterile sodium chloride in 20 experimental cases, wasdelivered upon the chorioallantoic membrane using aJencons (England) Sealpipette (0.5–10 ll) attached to amicromanipulator which was stabilized to a stereotacticframe. Immediately following, antibiotic (10,000 U/mlpenicillin G-10 mg/ml streptomycin sulfate, BiologicalIndustries Ltd., Beit Ha’emek, Israel) was injected inovo, and the shell window was re-inserted and its po-sition sealed with cellophane tape. Two other intactcontrols provided a comparison of developmental ageand days of incubation. Three additional cases in whichthe toxin was prematurely released upon the shell, wereadded to the control group. The operated eggs wereplaced in a horizontal position in the incubator, with-out further rotation, with adjacent eggs touching eachother so as to facilitate further embryonic development(Vince et al. 1976). Between chronologic day E20 and

Table 1 The effect of b-BT injection on geniculate, auditory and vestibular ganglia, and anterior mandibular taste structures

Case Chronologicagea

Developmentalageb

Bodilymovementat utilization

Geniculate ganglioncell counts:

Auditory andvestibularganglia

Numberof tastebudsf

Bud primordia:

Left side Right side Cellgroupingsf

Solitaryclear cellsf

B43 E20 E17 No 1 27 Absent 0 (0.0)g 13 (0.4) 8 (0.2)B75 E20 E17 No 20 42 Absent 0 (0.0) 10 (0.2) 18 (0.3)B70 E22c E21 No 3 9 Absent 0 (0.0) 4 (0.1) 26 (0.5)B72 E22c E21 No 0 5 Absent 0 (0.0) 9 (0.1) 19 (0.5)B81 E20 E19 Yes –e 281 Absent 50 (1.4) 0 (0.0) 0 (0.0)B224 E21 E21 Yes 2 125 Present 40 (1.8) 0 (0.0) 0 (0.0)B77 E22d E21 Yes 422 331 Present 37 (2.5) 0 (0.0) 0 (0.0)

aChronologic age = number of incubation days at utilizationbDevelopmental age at utilization as determined by nostril-to-upper beak, and third toe lengths [(Hamburger and Hamilton (1951),Ganchrow and Ganchrow (1987)]cThat is, the chick did not pip through the shell on the expected last gestation day of 21 incubation daysdPipping (cf.c)eCells not counted due to tissue staining artifactfBilateral countsgNumber in parenthesis = number of buds, bud primordia, or solitary clear cells/gland duct opening

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E22 (see Table 1), eggs were individually removedfrom the incubator, rapidly opened and the embryoremoved. Embryos were observed and recorded forspontaneous body movement and response to gentlebody pinch (Morris-Wiman et al. 1999), anesthetized(6% sodium pentobarbital, 0.05 ml, i.p.), their chestcavity opened to verify a heartbeat, and then decapi-tated. Antero-posterior incisions were made in thecheeks so as to expose lower beak and palatal tissue tosubsequent immersion in 10% formalin-saline for atleast 1 week. Subsequently, anterior lower beak andpalatal tissues were dissected and placed in fresh fixa-tive for at least 1 additional week. Also, followingremoval of the brain, the cranium was blocked coro-nally, anterior and posterior to the external auditorymeatus, so as to verify and analyze the geniculate,auditory and vestibular ganglia. When utilized, thedevelopmental age of control and experimental animalswas detemined by standard anterior edge of nostril-to-anterior edge of beak, and third toe length measure-ments (Hamburger and Hamilton 1951; Ganchrow andGanchrow 1987). This permitted determination of thedelay in development between toxin-injected, sham-control, and intact control cases.

Geniculate ganglion lesion (otocyst removal)

Geniculate and cochleo-vestibular ganglia are fused as acomplex attached to the medial side of the otocyst at E2.5 (Hamburger and Hamilton stages 14–16; Tello 1923;Romanoff 1960; Peusner and Morest 1977). Also, bystage 14, the embryo has turned to lie on its left side withthe hindbrain flexure at 90� to the forebrain, providing aview of the right side of the head. These stages are theleast compromising for attempting otocyst surgery, as byE3 (stages 17–19) the head has rotated antero-inferiorlyto lie in direct apposition to the heart (Hamburger andHamilton 1951).

Single eggs (n=145) were removed from the incuba-tor at E2.5, and the shell was windowed (�50 mm2).Visualizing at 40· magnification with the operationmicroscope, outer and inner shell membranes wereopened with #5 forceps, after which, in some instancesto better identify the ectodermal region of the otocyst, adrop of autoclaved, sterile 0.3% neutral red was appliedby forceps onto the embryo. A curved, sharpenedtungsten needle fused into a glass capillary pipette wasused to incise around and pluck out the otocyst (laterverified with the operation microscope). Immediatelyfollowing, antibiotic injection, and shell window re-insertion and sealing were carried out as described withb-BT injection cases. Eggs were returned to the incu-bator using the same conditions until utilization as withb-BT injection. The first posthatching day is designatedas H1. Control cases (n=69) included sham-operated(i.e, as described for otocyst removals but either notablating the otocyst, or only windowing the shell, orleaving the egg intact) embryos.

Histologic preparation and staining, and digital imaging

Dissected lower beak and palatal oral tissue, and thecranial tissue block containing geniculate, vestibular andauditory ganglia, were mildly decalcified (RDO, DupageKinetic Laboratories, Inc., Plainfield, IL, USA), paraffinor methacrylate embedded and serially sectioned at 8–10 lm (oral tissue) or 12 lm (cranial tissue). Oral tissuewas stained with Harris’ hematoxylin and eosin (H andE) such that any individual staining series containedslides from both anterior lower beak and palatal areas.Oral tissue in additional intact control cases was silver-stained [Bodian-Senn, and Fitzgerald stains, 14 lm; ra-pid Golgi (Morest modification, kindly provided by Prof.D. Kent Morest), 120 lm] for analysis of nerve fibers ingemmal and perigemmal tissue). Cranial sections wereMasson trichrome stained. Selected stained, or immu-noreacted sections, or photographic negatives (Zeiss MC63 camera / Standard 16 research microscope linkage)were scanned using a CCD camera (arcViewer version1.21, Baumer Optronic, Radeberg, Germany). Imageswere adjusted for contrast and brightness, labelled andassembled as plates using Adobe Photoshop 7.0.

Immunocytochemistry

Deparaffinized sections either were pre-treated withpronase (1:20) in the vimentin procedure, or microwaves(800 W, citrate buffer) in all other immunocytochemicalprocedures. Separate primary antibody incubations in-cluded one of the following: (1) mouse monoclonal, anti-intermediate filament protein, vimentin (purified frombovine lens), clone V3B4 [specificity: anti-chicken, anti-human (Bohn et al.1992); epitope: alpha-helical coil 2part of the vimentin rod domain; molecular weight:57 KDa, Progen (Heidelberg, Germany]; 1:100 dilution;(2) neuroactive marker, mouse monoclonal anti-chicken,neural cell adhesion molecule (NCAM), clone 5e, [epi-tope: extracellular domain; molecular weight: 180, 140,and 120 kDa (Frehlinger and Rutishauser 1986; Wa-tanabe et al. 1986), Developmental Studies HybridomaBank (University of Iowa, Iowa City, IA); supernatant,1:2 dilution]; (3) pan-neuronal marker, rabbit polyclonalanti-human, neuron-specific enolase (NSE), Medisera(Stockholm, Sweden); 1:1,000 dilution; (4) pan-neuronalmarker, rabbit polyclonal anti-human protein geneproduct 9.5 (PGP 9.5), Biotrend (Cologne, Germany);1:6,000 dilution. Primary antibody incubations werecarried out overnight at 4�C.

For secondary antibody incubations, after washing inPBS, the sections were reacted with either goat anti-mouse (vimentin, NCAM), or goat anti-rabbit (NSE,PGP 9.5) biotinylated IgG antibody (Dako, Copenha-gen, Denmark, diluted 1:400 in PBS) for 1 hour at roomtemperature. The reaction product was visualized byusing an avidin-biotin-peroxidase complex (Vectastain-Elite, Vector Labs, Burlingame, CA, USA) followed byincubation with 0.3% diaminobenzidine/H2O2.

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With all molecular markers, hematoxylin counter-stain was used on selected immunoreacted sections.

The following controls were carried out: (1) omissionof the primary antibody in order to rule out nonspecificbinding of the secondary antibody; and (2) in-parallelincubation of tissue (e.g., human, and rat tongue) pre-viously reported to be immunopositive for the markerstested.

Geniculate ganglion analysis

The intracranial topographic relations among thegeniculate ganglion, basilar papilla and auditory gan-glion are shown in a coronal section through the head ina 1-day-old hatchling (Fig. 1). Each serial section(12 lm) through the geniculate ganglion was drawnthrough a camera lucida attached to a Zeiss researchmicroscope (Neofluar lens, ·40; oculars, ·10) at a final

magnification of ·620. Only cell somata with completesomal and nuclear envelopes were selected and drawn.Nuclear diameter was determined as an average of nu-clear length as measured in both short and long axes.The number of ganglion cells was determined by theformula of Abercrombie, with correction for ‘split’ cellsin adjacent sections (Konigsmark 1970). In lightmicroscopic, Masson trichrome stained sections, intactand unoperated geniculate and vestibular ganglion so-mata are similar with respect to having a coarse, vacu-olated cytoplasm, whereas auditory ganglion somatahave a paler, smoother and finer grain cytoplasm (notshown). Therefore, neurons dispersed along the sensoryroot of the facial nerve and located between the genic-ulate ganglion laterally, and vestibular ganglion medi-ally, and whose somal staining properties are similar togeniculate or vestibular ganglion neurons, were notdrawn or counted for determining geniculate ganglioncell counts. The degree of geniculate ganglion damage inunilateral otocyst ablation cases was calculated as100%�[number of geniculate ganglion cells on theoperated side/number of geniculate ganglion cells on theunoperated side·100%].

Recorded observations also were made of the pres-ence or absence of the auditory and vestibular ganglia,basilar papilla, and columella.

Taste bud analysis

Each serial section (8–10 lm) through the anterior lowerbeak which contained possible taste bud primordia(solitary clear cells, or, small groupings of cells), or moredeveloped identifiable taste buds lying adjacent glandducts in epithelium was drawn through a camera lucida(see above). In following individual buds serially, thatsection through the greatest part of the taste bud wasselected for measuring bud width and vertical length,and characterizing gemmal shape. Early developingbuds have a relatively spherical shape (i.e., a gemmallength/width ratio=1.2), whereas the most mature budsare ovoidal (i.e., a gemmal length/width ratio=2.0), andintermediate spherical-ovoidal buds are between thesetwo ratios (Ganchrow and Ganchrow 1987, 1989a;Ganchrow et al. 1997). Gemmal counts are reported asboth total number of buds (as is the case in most tastebud studies), and, number of buds/gland duct opening(Ganchrow and Ganchrow 1987). This latter parametermore accurately reflects gemmal counts since in embryosthe number of taste buds and gland duct openings in thetaste bud region are positively correlated (Ganchrowand Ganchrow 1987).

Statistical analysis

The Mann–Whitney U test for independent samplescompared geniculate ganglion cell, or taste bud counts inb-BT-injected, or otocyst-lesioned groups versus controlgroup embryos. The Friedman two-way ANOVA test

Fig. 1 Masson trichrome-stained, coronal section through the headof a 1-day-old hatchling chick (Case 915), unoperated side. Lateral-to-medial relations are represented by up-to-down relations in thissection. Abbreviations: ag auditory ganglion, bp basilar papilla, ccolumella (lateral edge), eam external auditory meatus, gggeniculate ganglion, icb intracranial bone, me middle ear cavity, rsensory root of the facial nerve. Scale bar=300 lm

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for related samples compared geniculate ganglion cell,or taste bud counts, or gemmal width between operatedand unoperated sides across unilateral otocyst-lesionedcases. Individual otocyst-lesioned case analyses for theseparameters were tested by the Kruskal–Wallis one-wayANOVA (Siegel 1956). Since it was expected that b-BTinjections, or otocyst ablation probably would reducethe number of geniculate ganglion cells and/or tastebuds, an a priori one-tailed significance level of P £ 0.05was chosen.

Results

b-BT-injected cases

General observations

The behavioral effectiveness of ß-BT injection at utili-zation [developmental ages E17 (n=2), and E21 (n=2)]was evidenced by the absence of spontaneous grossmovement and absence of response to gentle body pinch(first four cases, Table 1; see also Morris-Wiman et al.1999), but a visible heartbeat upon opening the chestcavity. Further, absence of body movement also wasshown by an inability to pip through the shell on theexpected last gestation day (E21) such that the chicksometimes remained in ovo for 22 incubation days (E22)before utilization (n=2, Table 1). Cranial tissue, serialsection analyses showed the absence of auditory andvestibular ganglia (see Hirokawa 1977) and few, if any,remaining geniculate ganglion cells. Distal and/or prox-imal processes of remaining geniculate ganglion cellsusually were not discernible. In these cases, identifiedtaste buds were not evident. However, signs were evidentof possible early developing taste bud primordia (solitary

clear cells and/or small groupings of cells) adjacent glandducts in epithelium (n=4, Table 1). All of the above signscontrast with those in three additional cases (Table 1) inwhich toxin injection was less effective as evidenced bythe presence of body movement, and, auditory and ves-tibular ganglia at utilization [developmental ages E19(n=1) and E21 (n=2)]. Gustatory tissue analysis in theselatter cases showed a reduced number of taste buds/glandduct opening as well as geniculate ganglion cells (two ofthree cases) as compared with developmental age-mat-ched controls, and interestingly, the absence of signs oftaste bud primordia (three of three cases). In all seven b-BT-injected cases described above, some evidence of thebasilar papilla and columella was present.

Delay in development

Sixty percent (12 of 20 cases) of the b-BT-, and 62% (fiveof eight cases) of the sodium chloride-injected controlanimals survived until utilization. The second and thirdleftmost columns in Table 1 reveal the delay in devel-opmental age in b-BT-injected animals (n=7). As withcases undergoing unilateral otocyst removal (Table 2),the greater delay occurs in the chronologically youngeranimals: a slightly earlier utilization age (E20) usuallyindicates a developmental delay of 1–3 days, whereasembryos utilized while in ovo on the usual last gestationday (E21), or 1 day thereafter (E22), more closely matchtheir expected developmental age. This is supported bythree additional b-BT-injected cases (B55, B221, andB230, not shown) utilized between E19 and E20 whosedelay in developmental age was 1–4 days. Table 1 alsoshows that the absence of body movement, or auditoryand vestibular ganglia at utilization do not predict adelay in developmental age based on external body

Table 2 Effect of geniculate ganglion (Gg) lesion on anterior mandibular taste buds

Case Chronologic agea Developmental age Gg damageb Gg cell counts Number of taste buds

Operated Operated side Unoperated side Operated side Unoperated side

912 E20–21 E18 90% 43 444 39 (1.8)c 68 (1.9)c

916 H1 H1 85 77 510 26 (0.6) 51 (2.1)909 E20 E18 53 259 551 44 (1.5) 51 (2.0)914 E21–22 E20 33 273 410 14 (0.6) 31 (1.3)

Control Right side Left side

11d E18-19 E18-19 436 4258e E19 E19 566 57231d E20 E20-21 482 398B45f E20 E20-21 -g 523

aE embryonic (incubation) day; H posthatch day (i.e., the chick had pipped through the shell and was chirping) at utilizationbDegree of damage=100%�[number of geniculate ganglion cells on the operated side/number of geniculate ganglion cells on theunoperated side·100%] (rf. Materials and methods)cNumber in parenthesis = number of buds/gland duct openingdIntact, unopened eggeSham-operated: egg windowed and taped (rf. Materials and methods)fSaline injection administered onto the chorioallantoic membranegCells not counted due to tissue staining artifact

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features (Hamburger and Hamilton 1951; Ganchrowand Ganchrow 1987). In contrast, sodium chloride-in-jected (n=5), or intact (n=3) cases utilized at chrono-logic ages E18–E21 are matched for their expecteddevelopmental age (representative cases, Table 2).

The absence of gross body movement after b-BTinjection reported here is consistent with results previ-ously reported in embryonic chick (Hirokawa 1977,1978) and mouse (Morris-Wiman et al. 1999), and isindicative of the neurotoxic effect of b-BT.

Geniculate ganglion cells

Analysis of results in the first four cases listed in Table 1shows that a single b-BT injection administered ongestation day E12 significantly [U(7, 8)=0, P<0.000]reduced, bilaterally, the number of geniculate ganglioncells (range=0–42 cells/ganglion, developmental ag-es=E17 and E21) as compared with controls [Table 2:range=398–572cells/ganglion, developmental ag-es=E18–E21)]. Control counts reported here are con-sistent with those reported for the geniculate ganglion(range=353–475 cells/ganglion) in 20-week-old BrownLeghorn chickens (Gentle and Hunter 1987). Since theformulaic calculation (Konigsmark 1970) of ganglioncell counts includes the nuclear diameter of the ganglioncell, separate analysis of the effect of ß-BT injection onnuclear diameter indicated no significant [U(8,8)=20,P>0.05] difference between the aforementioned b-BT-injected (n=4) versus control cases.

Taste buds

Table 1 shows that b-BT injection in the first 4 caseslisted, resulted in no identifiable taste buds, bilaterally,in the anterior floor of the mouth at developmental agesE17 and E21, such that the mean number of taste buds/gland duct opening was significantly [U(4,5)=0,P<0.008] reduced as compared with controls (n=5,developmental ages E17 and E19–E21, cases not shown).This was demonstrated both in right [U(4,5)=0,P<0.008 and left [U(4,4)=0, P<0.014 (in one controlcase, the left side epithelium was not analyzed)] halves ofthe anterior floor of the mouth. Similarly in sampledpalatal tissue of these toxin-injected cases, identifiabletaste buds were absent. Examples of normally develop-ing taste buds as well as oral (anterior floor of themouth) tissue immunoreactivity (IR) to vimentin,NCAM, PGP and NSE are seen in Fig. 2. In contrast,the absence of identifiable taste buds as well as oral(palatal) tissue-IR to NCAM and vimentin after b-BTinjection are seen in Fig. 3.

Signs of putative gemmal primordia

In those four cases in which b-BT injection effectivelyresulted in absence of identifiable taste buds and nerve

fibers in anterior floor of the mouth (Table 1) and pal-atal epithelium, signs of possible early taste bud pri-mordia in both regions of oral epithelium were evidentas solitary clear cells, or small groupings of cells exhib-iting relatively clear cytoplasm. These putative pre-budstructures always were lying adjacent gland ducts inepithelium, a relationship that typically characterizestheir proximity as seen in normally developing buds (rf.Fig. 1; Ganchrow and Ganchrow 1987). The cells ofthese presumed taste bud primordia are usually verti-cally oriented in epithelium and characterized by clearcytoplasm as compared with adjacent epithelial, salivarygland, and subepithelial connective tissue cells. Someputative gemmal primordial cells are doubly nucleolated(Fig. 4). In these successful b-BT-injected cases, suchpre-bud signs in the anterior floor of the mouth wereevident bilaterally at E17 (solitary clear cells, range=8–18; small groupings of cells, range=10–13; n=2 cases),i.e., the earliest developmental age at which normallyearly developing spherical-shaped buds appear, and atE21 (solitary clear cells, range=19–26; small groupingsof cells, range=4–9; n=2 cases) when nearly the fullcomplement of mainly ovoidal-shaped buds normallyhas developed beyond the spherical-bud stage (Ganch-row and Ganchrow 1987).

Geniculate ganglion in surgically-lesioned otocyst cases

General observations

Five percent (7 of 145 animals) of the experimental casessurvived the necessarily long delay period between sur-gery on chronologic day E2.5 and utilization at devel-opmental ages E18-H1. Eighty-eight percent (127 of 145animals) of the cases died within one week, and 7% (11of 145 animals) after one week, postsurgery. This highattrition rate in long-term survival chick embryos afterearly otocyst (Parks 1979; von Bartheld et al. 1992), orrhombomere (Wahl and Noden 2001) ablation at E2.5–3(Hamburger and Hamilton stages 14–17) has been re-ported.

Table 2 indicates the effect of degree of unilateralgeniculate ganglion lesion on geniculate ganglion andanterior mandibular taste bud counts. Selected experi-mental cases (n=4, developmental ages E18, E20 andH1) are ordered by decreasing degree of geniculateganglion damage. Table 2 also shows the range ofgeniculate ganglion cell counts in controls that includeintact, sham-operated, or saline-injected cases (n=4,developmental ages E18–19–E20–21). It may be notedthat in intact control Case 31 there is a 17.5% differencein geniculate ganglion cell counts between both sides.With this difference as a baseline, two additionalexperimental cases at developmental ages E18 were ex-cluded from the analysis since geniculate ganglion cellcounts between both sides differed by 14 and 18%,respectively. Cranial tissue, serial section analysesshowed that in embryos sustaining 90 (Case 912), 85

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(Case 916), and 33% (Case 914) geniculate gangliondestruction (Table 2), the auditory ganglion and basilarpapilla were absent while the vestibular ganglion eitherwas absent or present to some extent. In Case 909 sus-taining 53% geniculate ganglion damage, auditory andvestibular ganglia and basilar papilla were present. Aswith b-BT-injected cases, the columella was present in allseven experimental cases (developmental ages E18, E20and E20–E21), similar to results in other otocyst abla-tion cases (Peusner and Morest 1977). Also compared tob-BT-injected cases (Table 1), the second and thirdleftmost columns in Table 2 reveal that delay in devel-opmental age was less severe though consistently greaterin E20 versus older utilized embryos.

Geniculate ganglion cells

Table 2 shows that the number of geniculate ganglioncells on the unoperated side (range=410–551 cells, n=4cases) in geniculate ganglion-lesioned cases is consistentwith, and not significantly different [U(4,7)=11,P>0.05] from that reported in controls (range=398–572 cells, n=4 cases), and adult chickens (Gentle andHunter 1987). In contrast, on the operated side, thedegree of geniculate ganglion damage ranged from 33–90%, in each case defined relative to the respectiveganglion cell count on the unoperated side (Table 2).Comparing across degree of ganglion damage, thereare significantly [vr

2 (1)=4, P<0.025] fewer geniculate

Fig. 2 Control cases B81 (a, e,f) and 231 (b, c, d,), both E19developmental age. Anteriorfloor of the mouth.Hematoxylin counterstain. aReference section in which theanti-PGP primary antibody hasbeen eliminated. Taste buds (Tupper borders demarcated bydots) are typically located oneither side of a salivary glandduct (L lumen of duct) openingintraorally. C subepithelialconnective tissue. b Arrowsindicate obliquely oriented,vimentin-positive (brown)gemmal cells, characteristic ofchick taste buds. c Taste bud(demarcated by dots). Arrowsindicate NCAM-IR in fiberslocated intragemmally, and insubgemmal connective tissue. dTaste bud (border demarcatedby dots). Arrows indicate PGP-IR (brown) in fibers locatedintragemmally and insubgemmal connective tissue. eNCAM-immunoreactivity (IR)(brown) particularly is evidentin subepithelial connectivetissue (C), but not epithelium(E), in this section. Adeveloping spherical-ovoidalshaped bud (upper borderdemarcated by dots) liesimmediately to the left of agland duct (L lumen of duct)opening intraorally. f NSE-positive (brown) fibers (arrows)within a nerve bundle in deepconnective tissue. Scale bar: thescale bar in f=70 lm, and asapplied to a and b=40 lm, cand d=30 lm, and e=100 lm

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ganglion cells on the operated versus unoperated side.Though three of four animals also showed a decrease innuclear diameter of the ganglion cell on the lesionedside, overall analysis indicated no significant difference[vr

2(1)=1, P>0.05 between operated and unoperatedsides (not shown).

Taste buds

In general, in the anterior floor of the mouth, embryossustaining unilateral geniculate ganglion damage atE2.5 incubation days showed a significant [vr

2(1)=4,P<0.025] decrease in the mean number of taste buds/

gland duct opening as compared with the unoperatedside (Table 2). Interestingly, with large geniculate gan-glion lesions, when only 10% (Case 912, E18, 90%ganglion damage) and 15% (Case 916, H1, 85% gan-glion damage) of the ipsilateral geniculate ganglion cellpopulation remained, respective gemmal counts showedthat as much as 57 and 51% of buds still were evidentcompared to the unoperated side (Table 2). This sug-gests that between developmental ages E18 and H1,sufficient innervation provided by �12% of survivinggeniculate ganglion neurons can maintain more thanone-half the total number of taste buds.

A corollary of the above is evident at developmentalage E18 with respect to the relation between the degree

Fig. 3 b-BT injected cases B75(a, d) (E17 developmental age),and, B72 (b, c) and B70 (e, f)(both E21 developmental age).Palate. Hematoxylincounterstain. a Arrows indicateabsence of taste buds inepithelium, on either side of agland duct (L lumen of duct)about to open intraorally. NoteNCAM-IR (brown) inconnective tissue (C), but notepithelium. b Absence of tastebuds on either side of a glandduct opening (hand symbol).Arrows indicate NCAM-IR inperiglandular (upper arrow) andperivascular (lower arrow)connective tissue. Epithelium(asterisk) in this section isNCAM-negative. c Arrowsindicate NCAM-IR in probableautonomic ganglion-like cellsadjacent an artery (A) in deepconnective tissue. d, e Arrowsindicate absence of taste buds,solitary clear cells, or smallgroupings of cells (cf. Fig. 4) inepithelium (E), on either side ofa gland duct (L lumen of duct),in sections tested for vimentin-IR. C, subepithelial connectivetissue. (f) Arrows indicateNCAM-IR fibers near andencircling an arteriole (A) indeep connective tissue. Scalebar: the scale bar in f=50 lm,and as applied to a andc=60 lm, b=220 lm, andd=100 lm

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of surviving geniculate ganglion cells and taste buds (rf.Table 2). Compared with Case 912 (10% survivingganglion cells, 57% surviving buds) above, in Case 909,47% surviving ganglion cells supported 86% of buds.That is, an �40% gain of surviving geniculate ganglionneurons resulted in a gain of �30% in the surviving budpopulation. By extrapolation, it would be expected thatbud counts at E18 would not significantly decrease with‡65% surviving ganglion cells. This is consistent withother otocyst-lesion data indicating that at E18, totalbud counts and number of buds/gland duct opening

between operated and unoperated sides do not essen-tially differ with 86% surviving ganglion cells (Case 910,not shown). These results also are supported by thecontrol baseline difference of 17.5% in geniculate gan-glion cell counts between both sides (Case 31, Table 2).

Taste bud differentiation

Of the geniculate ganglion lesioned cases listed inTable 2, comparisons of bud shape on the unoperated

Fig. 4 Examples of possible taste bud primordia (solitary clearcells, and, small groupings of cells) in the anterior floor of themouth (a, b, c; hematoxylin and eosin stain) and palate (d, e;hematoxylin counterstain), following b-BT injections in Cases B75(b) and B43 (d) (both E17 developmental age), and, B70 (a, e) andB72 (c) (both E21 developmental age). In all four cases there wasno evidence of identified taste buds after toxin injection in ovo (rf.Table 1). a Arrow indicates a vertically-oriented solitary cell inepithelium adjacent a salivary gland duct (cut obliquely; L lumen ofduct) about to open intraorally. The relatively clear cytoplasm ofthe solitary cell distinguishes it from nearby epithelial, gland, andsubepithelial cells. b Arrow in epithelium indicates two vertically-oriented cells with clear cytoplasm, to the left of a gland duct (notseen) which opened intraorally in an adjacent section. Note theclear cell at the left has two nucleoli. c Arrow in epithelium indicatesa cluster of cells with clear cytoplasm. Note the two central cells in

this cluster each have two nucleoli. Debris (D) fills the lumen of anadjacent salivary gland duct opening intraorally. d Arrow indicatesan oblique vertically-oriented, vimentin-positive (brown) solitarycell with relatively clear cytoplasm in epithelium, located adjacentand above a subepithelial salivary gland (cut obliquely; L lumen ofgland duct). Vimentin-IR also is evident in fibrocytes of subep-ithelial connective tissue. e Arrowhead (top left) indicates agrouping of two ovoidally-shaped and vertically oriented cells inepithelium displaying relatively clear cytoplasm. These clear cells lieadjacent salivary gland tissue (gland duct not seen in this section,and appear morphologically distinct from the darkly-nucleatedgland cells (arrow, lower right). Only periglandular connectivetissue (C) is NCAM-positive (brown). Scale bar: the scale bar ine=50 lm and as applied to a=45 lm, b and d=30 lm, andc=50 lm

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side at developmental ages E18 (n=2 cases) and E20(n=1 case) indicate the case-specific nature of normalgemmal differentiation. For example, at E18, respectivepercentages of progressively developing spherical,spherical-ovoidal, and ovoidal buds are 49, 46, and 6%(n=68 total buds) in Case 912, as compared with 86, 12,and 2% (n=51 total buds) in Case 909. Interestingly, onthe operated side in these cases, maintaining a compa-rable unoperated stage of differentiation (see above) insurviving buds reflects the percentage of survivinggeniculate ganglion cells: Case 912 (10% survivingganglion cells) showed 74% spherical, 23% spherical-ovoidal, and 35% ovoidal buds (n=39 total buds),whereas Case 909 (47% surviving ganglion cells) showed77% spherical, 20% spherical-ovoidal, and 2% ovoidalbuds (n=44 total buds) ovoidal buds. That is to say, inearly bud development (E18), almost one-half of thesurviving geniculate ganglion population can more clo-sely maintain the proportion of surviving buds at thesame developmental stage as on the unoperated side.However, in the later perihatching period, the propor-tional stages of differentiation in surviving buds do notsimply reflect the percentage of surviving ganglion cells:at E20 (Case 914: 67% surviving ganglion cells), therewas a greater proportion of earlier-stage spherical budson the operated (60%) versus unoperated (48%) sides;and a reduced proportion of later-stage, spherical-ovoi-dal buds on operated (31%) versus unoperated (52%)sides. In contrast, at H1 (Case 916: 15% survivingganglion cells), there was a smaller percentage ofspherical buds on operated (55%) versus unoperated(82%) sides, yet an additional 33% of surviving buds hadreached the ovoidal stage of gemmal development. Re-sults in these latter two cases were unexpected since theircalculated geniculate ganglion/taste bud innervationratios (i.e, number of ganglion cells/bud) were similarlyabout two times greater on the operated versus unop-erated sides. This suggests that after E18, the survival ofdeveloping buds including their stage of differentiationprobably reflect their degree of innervation, and rate ofdifferentiation.

Taste bud width

Notwithstanding degree of geniculate ganglion damage,comparisons of gemmal width in spherical-shaped budsat E18 indicate that geniculate ganglion lesion per seresults in a significant decrease in ipsilateral sphericalbud width compared with the unoperated side {[Case912: 40.0±1.6 lm, operated side versus 47.0±1.8 lm,unoperated side; (vr

2(1)=25.34, P<0.0005)]; [Case 909:44.4±1.9 lm, operated side versus 47.0±1.6 lm, un-operated side; (vr

2(1)=22.68, P<0.0005)]}. While thissuggests that there may be fewer bud cells/surviving bud,geniculate ganglion cell nuclear diameter in these casesalso decreased on the lesioned side implying that gan-glion cell somal size had shrunk as well, i.e., a smaller

ganglion cell was innervating a bud possibly containingthe same number of smaller albeit shrunken cells.

Although a similar trend in decreased diameter ofspherical buds is noted at E20 (Case 914: budwidth=35.9±1.5 lm, operated side versus 39.5±lm,unoperated side), the difference is not significant[vr

2(1)=2.02, P>0.05] between lesioned and unlesionedsides, in part perhaps due to the relatively small numberof buds observed in this embryo. For technical reasons,bud width at H1 (Case 916) was not analyzed.

Taste bud innervation

Since silver-stained Case 912 (E18) sustained the greatestgeniculate ganglion damage (Table 2) and exhibitedprominent changes in bud differentiation (see above),this case also was analyzed for presence of intragemmalinnervation in spherical-shaped buds. On the unoperatedside, 23% (16 of 68 buds) of buds contained evidence ofintragemmal fibers, and 56% (9 of 16 buds) of these werespherical-shaped buds. Of those buds lacking signs ofintragemmal fibers, 38% (20 of 52 buds) of buds showedsigns of perigemmal or immediately deep subgemmalfibers. Though fewer total buds appeared on the lesionedside, 15% (6 of 39 buds) still showed evidence of intra-gemmal fibers and all of these buds were of sphericalshape. Of those buds on the lesioned side lacking signsof intragemmal fibers, 21% (7 of 33) of buds showedsigns of perigemmal or subgemmal fibers. Thus, atdevelopmental age E18, at least some early-appearingspherical-shaped buds contain intragemmal (presumablygustatory) fibers. Additional support for evidence ofintragemmal fibers in spherical-shaped buds at E18 wasalso recorded in intact, silver-stained Case 871 (notshown).

Discussion

Taste bud primordia and early-developing buds:the issue of nerve-dependence in b-BT-injected cases

b-BT injection at E12, in ovo, severely damaged theperipheral taste system at developmental ages E17 andE21, reflecting part of a molar cranial sensory and so-matomotor effect initiated by the neurotoxin. The mosteffective ß-BT injections destroyed 91–100% of the ex-pected number of geniculate ganglion neurons in controlcases (cf. first 4 cases, Table 1 versus 4 control cases,Table 2). While there were neither identifiable taste budsnor NCAM- or PGP 9.5-immunopositive fibers in oralepithelium, putative taste bud primordia persisted inepithelium adjacent to salivary gland duct openings,where normally developing taste buds would have ap-peared. This suggests that in the avian chick, (1) gemmalprimordia may be taste nerve-independent which sup-ports several studies in fish, amphibian and rodential

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mammal (see Introduction); and (2) there may be pre-programmed (competent) regions of oral epithelium inwhich gemmal primordia can develop, perhaps inducedby adjacent endoderm (Begbie et al. 1999). While argu-ably some of the putative gemmal primordial structurescould be atrophic remnants of buds deprived of theirgustatory (chorda tympani nerve) innervation (Ganch-row and Ganchrow 1989b), (1) these putative progenitorcells appeared within an otherwise healthy-appearingepithelium (see also Fig. 5d, Morris-Wiman et al. 1999);and (2) b-BT administered at E12 probably effectivelydestroyed chorda tympani nerve axons well in advanceof their possible neural invasion of epithelium at orpreceding E17 (see below). This is consistent with resultshere in ß-BT-injected cases showing that NCAM im-munopositivity was evident in nerve fibers and otherstructures in connective tissue but not epithelium.

Intriguingly, these putative gemmal primordia werestill observed at E21 (as well as E17), when normallymost gemmal structures are forming, or have alreadyattained, a mature ovoidal shape (Ganchrow andGanchrow 1987, 1989a; Ganchrow et al. 1995, 1997).This suggests that while gemmal primordia may benerve-independent, they may tropically direct that gus-tatory sensory innervation necessary to further gemmaldifferentiation [cf. Oakley 1991, Krimm and Hill 2000;Mistretta and Hill 2003; Oakley and Witt 2005; see alsoin chick embryonic cultures: developing cochleo-vestib-ular ganglion neuritic outgrowth to otic epithelium (Ardet al. 1985; Hemond and Morest 1992)]. Hence, after ß-BT-induced damage, any nerve types that survived wereinadequate to promote autocrine- and/or nerve-inducedtaste bud cell differentiation and gemmal maturation.This is supported by results with less damaging ß-BTinjections (last 3 cases, Table 1) that showed a greaternumber of geniculate ganglion neurons and identifiabletaste buds, and absence of gemmal primordia, implyingthere was a sufficient number of surviving neurons tofurther bud development beyond the bud primordiumstage.

The process initiating gemmal cell and taste budmaturation probably is rapid and closely timed as indi-cated by previous ultrastructural (Ganchrow andGanchrow 1989a) evidence, and silver-stained materialpresented here, showing that intragemmal fibers alreadyare evident at E18 when spherical-shaped buds pre-dominate. Additional silver-stained oral tissue in intactchick embryos (Ganchrow and Ganchrow, unpublisheddata) indicates that fibers of unknown origin have pen-etrated the 2–3 thick oral epithelium cell layer atdevelopmental ages E15–E17, and are evident in some,but not all, putative taste bud primordia at develop-mental ages E16–E17. Thus, in the normally developingchick there probably is a very narrow window of timewithin which taste bud primordia establish contact withgeniculate ganglion cell axons. For example, in thehuman at 8 weeks postovulatory age, ultrastructuralevidence shows that the earliest gemmal primordiaalready exhibit putative afferent synaptic contact with

intragemmal nerve fibers (Witt and Reutter 1996).Similarly, in the chick auditory system, identifiableauditory hair cells in basilar papilla are first seen atE8–E9 in parallel with initial signs of cochlear nerve-haircell synaptogenesis (Whitehead and Morest 1985a, b;cf. Hirokawa 1977).

It has been argued that in young postnatal rat oralepithelium, solitary ‘chemosensory’ cells precede andperhaps tropically mark the location of later-appearing,identifiable taste buds in taste bud-bearing papillae (re-view: Sbarbati and Osculati 2005). Whether the solitaryclear cells or groupings of cells seen here in ß-BT-in-jected cases are functionally chemosensory await furtherinvestigation (cf. fish and amphibians: Whitear 1992;Finger 1997; Kotrschal et al. 1998; Sbarbati et al. 1999;Sbarbati and Osculati 2005, Fishelson 2005; rodents:Finger et al. 2003).

Surgically-lesioned (otocyst ablations) geniculateganglion and the relation to taste bud developmentand survival

The effects of early embryonic otocyst ablation (E2.5)upon gemmal development (E18-H1) reported here areconsistent with, and support the thesis that taste budmaturation, maintenance, and survival are nerve-dependent (see Introduction). Lesioned case analysesindicate that (1) overall decrease in the number ofidentifiable taste buds appears to be dependent on theloss of adequate geniculate ganglion innervation; (2)sufficient innervation apparently provided by �12% ofgeniculate ganglion neurons can maintain more than50% of the total number of taste buds; (3) in early buddevelopment (E18), (a) there is a direct relation betweenthe degree of surviving geniculate ganglion cells andtaste buds; and (b) almost one-half of the survivinggeniculate ganglion population can closely maintain theproportion of surviving buds at the same stage of gem-mal differentiation as on the unoperated side; (4) in thelater perihatching period (E20–H1) of bud development,proportional stages of differentiation in surviving budsprobably reflect their degree of innervation as well asrate of differentiation; and (5) irrespective of degree ofgeniculate ganglion damage, the proportion of survivingbuds can be sustained at the same differentiated budstage as on the unoperated side, or differentiate to a latergemmal stage.

Compared with toxin injection, focal geniculateganglion damage by means of otocyst lesion at E2.5more specifically was directed to interfering with earlyoutgrowth of the ganglion cells’ distal processes well inadvance of the appearance of incipient spherical tastebuds at E17 (Ganchrow and Ganchrow 1987). Further,it was expected that due to the relatively long 2.5–3 weeksurvival period between otocyst lesion and utilization,most dying geniculate ganglion cells would have beenphagocytized. Since the chick geniculate ganglion neu-ron population is finally postmitotic only by E5

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(D’Amico-Martel 1982), and is derived from the pha-ryngeal endoderm-induced (Begbie et al. 1999), firstepibranchial placode (Yntema 1937, 1944; D’Amico-Martel and Noden 1983; cf. Northcutt 2004) situatedimmediately anteroinferior to the otocyst, it is possiblethat some geniculate ganglion cells identified betweendevelopmental ages E18 and H1 were born between E2.5and E5. This is particularly relevant in those otocyst-lesioned cases that sustained 90 (Case 912) and 85%(Case 916) (Table 2) geniculate ganglion damage. Whe-ther these placodally-derived neuroblasts would havesuccessfully migrated to the location of the (geniculo-)cochleo-vestibular ganglionic mass or partially lesionedotocyst is not easily known (von Bartheld et al. 1992).For example, after ablation of single rhombomeres 2, 3or 4 in chick embryo at E2.5–3 (Hamburger–Hamiltonstages 14–17), placodally-derived trigeminal neuroblastsmay fail to migrate proximally to merge with neuralcrest derived neuroblasts in the developing sensory tri-geminal ganglion, or may form ectopic distal ganglia(Wahl and Noden 2001).

In general, between developmental ages E18 and H1,33–90% geniculate ganglion damage after unilateralotocyst lesion at E2.5 (Hamburger-Hamilton stages 14–16) results in a significant decrease in the mean numberof anterior mandibular taste buds/gland duct opening aswell as percentage of buds characterized by gemmalshape, as compared with the unoperated side in theanterior floor of the mandible. Notwithstanding naturalcell death possibly occurring on the operated side, asreported in normally-developing taste buds between E19and H1 (Ganchrow and Ganchrow 1987), more thanone-half of the surviving gemmal population can besustained by a relatively small percentage (�12%) of thesurviving geniculate ganglion cell population. Based onprevious studies in chicken (Saito 1966; Gentle andHunter 1983; Gentle and Clark 1985; Ganchrow andGanchrow 1985), it is estimated that normally eachanterior lower beak taste bud may receive up to ten(geniculate ganglion cell axonal) branches of the de-ef-ferented chorda tympani nerve (Gentle and Clark 1985;Ganchrow and Ganchrow 1989a). A comparable esti-mate in the rat suggests that each fungiform taste budnormally may receive �6 de-efferented chorda tympanibranches (Miller 1974; Miller and Preslar 1975; Farb-man and Hellekant 1978; Miller and Spangler 1982;Miller et al. 1978). The tastant-stimulated receptive fieldof rat, single chorda tympani axons includes an averageof two fungiform taste bud-bearing papillae (range=1–4papillae, n=9 fibers) (Miller 1971). The survival seenhere of a substantially large proportion of taste buds bya small proportion of geniculate ganglion neurons, couldbe accounted for by (1) autocrine-induced neuriticbranching in surviving geniculate ganglion cells as trig-gered by the ganglionic lesion at E2.5 and/or (2) axonalbranching tropically induced by surviving gemmal pri-mordia and early-stage spherical buds, beginning atE16–E17. In the latter case, the peripheral taste tar-get also could tropically direct the degree of innervation.

NCAM and BDNF are molecular factors possiblymediating these processes.

NCAM and BDNF: possible roles in peripheral tastesystem development

Cell surface glycoprotein, NCAM, is a member of theimmunoglobulin superfamily, and may have a role ingustatory development (review: Ganchrow 2000). In thedeveloping auditory system of chick, NCAM-IR ispresent in cochlear hair cell precursors and differentiat-ing hair cells, as well as distal and proximal processes ofcochlear ganglion cells, from E3 to E5 (i.e., initialinvasion of basilar papilla epithelium) to at least E17(i.e., hair cell synaptogenesis) (Hrynkow et al. 1998). Indeveloping fungiform and circumvallate papillae ofembryonic and newborn (from E16 through postnatalday 1) mouse, NCAM-IR already is evident in pre-tastebud dorsal epithelium, and continues to be presentspecifically within differentiating taste bud cells from atleast postnatal days 1–10.5 (Nolte and Martini 1992;Takeda et al. 1992; Miura et al. 2005). Interestingly,98% of these NCAM-immunopositive cells co-localizewith Mash 1 mRNA, a transcription factor possiblyinvolved in gemmal cell differentiation (Miura et al.2005). In the normal H1 chick when the adult comple-ment of taste buds is fairly well established, NCAM-IRis evident in gemmal cells as well as intragemmal fibers(Tabib 2000). As reported here in control cases at E19,NCAM-IR convincingly is seen only in intragemmal fi-bers and not gemmal cells. NCAM-IR also was evidentin periglandular and perivascular nerve fibers deepwithin connective tissue, in b-BT-injected chicks exhib-iting possible gemmal primordia at developmental agesE17 and E21. In the somatomotor system, ectopicexpression of NCAM in skeletal-specific muscle oftransgenic mice is reflected by significant increases in thenumber of terminal and preterminal sprouts as well aslength of presynaptic terminals in the neurmomuscularjunction suggesting, at least in part, an NCAM-medi-ated tropic effect of the target muscle upon its source ofinnervation (Walsh et al. 2000). In chick, it is not knownwhether NCAM is present at the period circumscribingearly gemmal development, i.e., localized in cells or fi-bers in peri-gland duct epithelium, or taste bud pri-mordia at E16/17, which would suggest autocrine cell-adhesive, tropic, and/or trophic roles of NCAM ingemmal development.

Neuronal growth factor, BDNF significantly in-creases regenerative neuritic robustness of outgrowthand length in cultured E12-E14 geniculate ganglion cellsof rat (Rochlin et al. 2000; Yamout et al. 2005). Also,BDNF receptor, TrkB mRNA and protein are present inembryonic and young adult geniculate ganglion cells ofrodents (e.g., Yamout et al. 2005). Hence in the presentstudy, BDNF may have contributed to furthering pro-posed neuritic branching of surviving geniculate gan-glion cells. Also, in developing peripheral taste tissue of

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rodent and human embryo, BDNF mRNA is evident inincipient fungiform and circumvallate papillae epithe-lium prior to and pari passu appearance of their residentgemmal primordia (Nosrat and Olson 1995; Nosrat et al.1996, 2000, 2001; Chou et al. 2001); immunocytochem-ical analysis shows that BDNF-IR occurs in taste budprimordia in circumvallate papillae of postnatal day 1mice (Uchida et al. 2003). In BDNF null mutant mice,50% of the geniculate ganglion cell population survivedyet could not rescue 60% of their gemmal targets intaste-bud bearing fungiform papillae (Mistretta et al.1999). In reviewing these results, Mistretta and Hill(2003) proposed a model for initial bud developmentwherein the immature bud (primordium?) expressed theBDNF neurotrophin and thereby would provide a‘‘survival factor for (innervating) neurons’’ (p. 773). Thisis consistent with studies in BDNF-overexpressing micein which, in the absence or reduction of the number offungiform taste buds, some chorda tympani axonsinappropriately remained below epithelium or appearedto innervate BDNF-overexpressing, non-gustatory fili-form papilla epithelium (e.g., Krimm et al. 2001).

It is not known whether BDNF in chick embryo islocalized to geniculate ganglion axons, early pre-budepithelium, or gemmal primordia so as to provide anautocrine mechanism for axonal outgrowth and buddevelopment as well as tropically contribute to genicu-late ganglion neuron survival. On the other hand,vimentin and cytokeratins 19 and 20 immunopositivityin gemmal primordia (e.g., solitary clear cells) of chickand human embryos (Witt and Kasper 1999; Witt et al.2000, and this report) suggests the importance of theseintermediate filament proteins to incipient bud devel-opment. Planned developmental studies in chick embryowill investigate the earliest times at which these proteins,as well as the G-protein, a-gustducin (in mice and rats:Finger et al. 2003), and calcium-binding protein, calre-tinin (in salamander and catfish: Barlow et al. 1996;Northcutt 2004, 2005) may be identified in pre-taste budepithelium, gemmal primordia and identifiable buds.Intriguingly, since chick gustatory, auditory and ves-tibular ganglia develop from a common ganglionic an-lage, molecular markers in-common may be indicativeof receptor cell development and survival in theirrespective peripheral sensory organs in chicks, and ver-tebrates in general.

Taste bud-salivary gland relations

Since taste buds in the avian chick are not papilla-borne,but rather, characteristically maintain an adjacent rela-tion in epithelium to salivary gland ducts which openintraorally, the possible close temporal dependence ofdeveloping buds on their papillary compartment(Farbman and Mbiene 1991; Whitehead and Kachele1994; cf. Witt and Reutter 1996, 1998; Mbiene andRoberts 2003) as affected by b-BT does not apply here.Indeed, while b-BT injection did not prevent continuing

development and appearance of gland ducts in oralepithelium of chick embryos (our unpublished observa-tions; see also Morris-Wiman et al. 1999), anteriortongue fungiform, but not posterior tongue circumval-late papillae in mice embryos were absent after similarinjection (Morris-Wiman et al. 1999). Also, since inchick, initial signs in mandibular epithelium of devel-oping downgrowths of salivary gland tissue towardsubepithelial mesenchyme [beginning at E8 (Ganchrowand Ganchrow 1987)] precede that of gemmal (begin-ning at E17) development by 9 days, it would seem thatthe origin of chick buds is probably not salivary gland-dependent. This is supported by our observations incontrol as well as ß-BT-injected chicks of E17 develop-mental age where residual salivary gland tissue is evidenton the surface of oral epithelium and continuous withdeeper-lying gland cells lining the gland duct lumen.These results, however, do not negate the possibility thatearly gemmal, and salivary gland development is medi-ated by independent epithelial-mesenchymal molecularsignalling (reviews: Cutler and Gremski 1991; Dennyet al. 1997).

Acknowledgments The authors would like to thank the KibbutzTzuba hatchery (Jerusalem) for their kindness in supplying theeggs. Also, we thank Ms Dita Goldstein, Ms Liah Bar-Dov, andMr Milu Sadovnic for carefully preparing histologic materials,Mrs. Sylvia Bramke for carefully carrying out immunohistochem-ical reactions, and Dr. Victor Belkin for meticulous care in helpingprepare digital images. A special thanks to Dr Kent Morest forproviding access to invaluable chick embryologic materials, and,Drs Kenna Peusner and Mark Whitehead for respectively sharingtheir expertise in in ovo procedure and demonstrating the fineraspects of otocyst surgery. The authors also wish to thank theanonymous reviewers for their cogent and thoughtful comments.

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