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In mice lacking functional brain-derived neurotrophic factor (BDNF), the number of geniculate ganglion neurons, which innervate taste buds, is reduced by one-half. Here, we determined how and when BDNF regulates the number of neurons in the developing geniculate ganglion. The loss of geniculate neurons begins at embryonic day 13.5 (E13.5) and continues until E18.5 in BDNF-null mice. Neuronal loss in BDNF-null mice was prevented by removal of the pro-apoptotic gene Bax. Thus, BDNF regulates embryonic geniculate neuronal number by preventing cell death rather than promoting cell proliferation. The number of neurofilament-positive neurons expressing activated caspase-3 increased on E13.5 in bdnf −/− mice, compared to wild-type mice, demonstrating that differentiated neurons were dying. The axons of geniculate neurons approach their target cells, the fungiform papillae, beginning on E13.5, at which time we found robust BDNFLacZ expression in these targets. Altogether, our findings establish that BDNF produced in peripheral target cells regulates the survival of early geniculate neurons by inhibiting cell death of differentiated neurons on E13.5 of development. Thus, BDNF acts as a classic target-derived growth factor in the developing taste system.
A small cranial sensory ganglion called the geniculate ganglion innervates chemosensory receptors responsible for the sense of taste that are on the front two-thirds of the tongue, the soft palate and the nasoincisor ducts of the hard palate. These neurons carry information from peripheral chemosensory cells to the central nervous system. The development of peripheral circuitry in the taste system is highly regulated, in that each taste bud is innervated by a specific number of neurons (Krimm and Hill, 1998; Zaidi and Whitehead, 2006). The mechanisms underlying this numerical precision are unclear, but the final number of taste neurons available to innervate each taste bud is determined by the relative contributions of cell proliferation, differentiation, and cell death within the geniculate ganglion during development. Early in geniculate ganglion development, cells are added rapidly (Altman and Bayer, 1982). By embryonic day 14.5 (E14.5) in rat (around E12.5–E13 in mouse), cell proliferation has slowed and thereafter the total number of neurons remains constant throughout the embryonic period (Carr et al., 2005). Like most regions of the nervous system, the geniculate ganglion overproduces neurons and undergoes a period of cell death, the peak of which is approximately E16.5 in rat (approximately E14–E14.5 in mouse) (Carr et al., 2005). However, total neuron number doesn’t change after E13.5 in rat indicating that neuronal proliferation and differentiation progress at equivalent rates through out much of embryonic development (Carr et al., 2005). A host of factors including neurotrophins likely regulate neuronal proliferation, differentiation, and death in the geniculate ganglion.
In mammals, the neurotrophin family consists of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT-4) (Huang and Reichardt, 2001). These neurotrophins are sometimes thought to be produced by peripheral target tissues, where they influence neuron number by preventing the death of ganglion neurons that innervate them (Huang and Reichardt, 2001; Vogel and Davies, 1991). Alternatively, they are sometimes expressed within sensory ganglia and along projection pathways where they influence neuron development before target innervation (Farinas et al., 1996). Neurotrophins not only regulate neuronal numbers by influencing survival of differentiated neurons, but can also influence survival, differentiation, and proliferation of neuronal precursors during early development (Ernfors, 2001; Farinas et al., 2002; Liebl et al., 2000; Lopez-Sanchez and Frade, 2002).
The final number of neurons that comprise the geniculate ganglion is influenced by BDNF. Relative to wild-type animals, bdnf −/− mice show a significant reduction in neurons in the geniculate ganglion by birth (Conover et al., 1995; Liu et al., 1995). Approximate 45–65% of the taste buds on the anterior tongue, whose survival requires gustatory innervation, are also lost in bdnf −/− mice (Mistretta et al., 1999; Nosrat et al., 1997; Sun and Oakley, 2002), indicating that many of the missing neurons are gustatory. The mechanism employed by BDNF during embryogenesis to influence the neuronal numbers within the adult geniculate ganglion is unknown. Since BDNF is expressed in the geniculate ganglion, tongue muscle, and gustatory targets of geniculate neurons (Nosrat, 1998; Nosrat et al., 1996; Nosrat and Olson, 1995; Schecterson and Bothwell, 1992), removal of BDNF from multiple locations, at any embryonic age and through a variety of mechanisms could result in a reduction of geniculate neurons.
In this study, we sought to determine how and when BDNF regulates the final number of sensory neurons that comprise the geniculate ganglion. We established that BDNF expressed in taste epithelium promotes survival of differentiated gustatory neurons as they first innervate their targets, before the peak of naturally occurring cell death.
All BDNF mutant (bdnf −/−) and wild-type mice were on a C57BL/6J background strain. Homozygous bdnf −/− embryos were obtained by breeding mice with heterozygous targeted mutations of the bdnf gene. Heterozygous bdnf −/+ mice were acquired from Jackson Laboratories (Bar Harbor, Maine, USA; #002266). Animals were genotyped by polymerase chain reaction, as described in protocols provided by Jackson Laboratories, http://jaxmice.jax.org/). Bdnf-lacZ mice were a kind gift from Kevin Jones (Dept. of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, USA) (Baquet et al., 2005; Gorski et al., 2003a; Gorski et al., 2003b). Bdnf-lacZ mice have been used successfully to examine BDNF-producing locations in the adult taste system (Yee et al., 2003). Animals were genotyped by polymerase chain reaction using the lacZ forward primer 5’-TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA-3’, and the lacZ reverse primer 5’-ATG TGA GCG AGT AAC AAC CCG TCG GAT TCT-3’. Embryonic mice were obtained from time-bred females that were placed with males just before the 8-h dark period and examined for plugs the following morning. The day a plug was observed was designated embryonic day 0.5 (E0.5). Heads from wild-type, bdnf −/−, bax−/−, and bdnf −/−/bax−/− double knockout mice at birth were a kind gift from David M. Katz (Dept. of Neurosciences, Case Western Reserve University, Cleveland, OH, USA).
Embryos aged 14.5 (E14.5) and older were immediately perfused using chilled 4% paraformaldehyde (PFA), and animals E13.5 and younger were fixed by immersion in PFA. All embryos were fixed overnight in 4% PFA. Following fixation, embryo heads were moved to 70% ethanol and processed for paraffin embedding. Immuno-detection of the cytoskeletal element class III β-tubulin (β-III tubulin) was used to visualize geniculate ganglion neurons. Serial transverse sections (5 µm) of paraffin-embedded embryo heads were collected on Fischer SuperFrost/Plus slides (Fisher Scientific, Pittsburgh, PA, USA). Paraffin was removed by immersion in CitriSolv (Fisher). Sectioned tissues were rehydrated in a graded series of alcohols, followed by 0.1 M phosphate-buffer saline (PBS), pH 7.4. Endogenous peroxidase activity was quenched by treatment for 15 min in a solution of 10% methanol/3% H2O2 in PBS. Slides were washed in dH2O (3 × 5 min each). In the antigen retrieval step that followed, slides were boiled in the microwave for 5 × 2min in citrate buffer (0.1 M citric acid/0.1 M Na citrate/dH2O; pH 6) with a 5 min rest between the two boiling periods, and then cooled down to room temperature. The sections were washed in PBS and blocked for 30 min in blocking buffer (PBS/5% goat serum/0.25% Triton X-100) and were then incubated overnight at 4°C in blocking buffer containing a 1:1000 dilution of monoclonal mouse anti-β-III tubulin antibody (TUJ1; Covance, Princeton, NJ, USA; catalog # MMS-435P). On the following day, the sections were washed in PBS (3 × 5 min) and incubated for 2 h at room temperature in blocking buffer containing a 1:200 dilution of biotinylated anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA; #BA-2000), and visualized with an ABC diaminobenzidine reaction. The following numbers of mice embryos were used for quantification: wild-type (n = 3 for E12.5, E14.5, E16.5, E18.5; n = 5 for E13.5); bdnf −/− (n = 3 for E12.5, E13.5, E16.5, E18.5; n = 4 for E14.5).
Pregnant females were injected 2 h before sacrifice with 50 mg/kg (intraperitoneal) of 2′-bromo-5′-deoxyuridine (BrdU; 5 ml/kg of a 10 mg/ml stock solution in 0.1 M Tris–HCl buffer, pH 7.5). Embryos were immersionfixed overnight in 4% PFA in PBS, pH 7.4. They were then placed in 30% sucrose/PBS overnight for cryoprotection, and frozen on Dry Ice in OCT embedding medium. Embedded embryos heads were sectioned transversely (16-µm), mounted on SuperFrost/Plus slides (Fisher), and allowed to air-dry. Fixed, mounted transverse sections were treated with 2 N HCl and heated to 37°C for 30 min. Slides were washed in dH2O (3 × 5 min each). In the antigen retrieval step that followed, slides were boiled for 15 min in citrate buffer (0.1 M citric acid/0.1 M Na citrate/dH2O; pH 6), and allowed to cool down to room temperature. Next, the slides were washed in PBS (3 × 5 min) and blocked in blocking buffer (described above) for 30 min. Sections were incubated overnight in the following primary antibodies: anti-BrdU (1:200, Accurate Chemicals, Westbury, NY, USA; #OBT0030), mouse anti-neurofilament (1:200, Millipore, Billerica, MA, USA; #MAB5266), and rabbit anti-cleaved caspase-3 (Asp175) (1:200, Cell Signaling, Beverly, MA, USA; #9661). The slides were washed in PBS (3 × 5 min), and then incubated for 2 h at room temperature in the following secondary antibodies: alexa 488 anti-rat (A11006), alexa 546 anti-mouse (A11030), and alexa-647 anti-rabbit (A21244;Invitrogen, Carlsbad, CA, USA) at a dilution of 1:200 in blocking buffer. Slides were dehydrated in a graded series of alcohols and cover slipped in CitriSolv with DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA). Immuno-labeled tissue was visualized using a fluorescence confocal microscope. Antigen-positive cells were quantified from n=3 each wild-type and bdnf −/− embryos.
Detection of β-Gal was performed either in whole-mount embryo tissue and in both sagittal and transverse sections. The embryos were immersion-fixed in ice cold 0.5% glutaraldehyde in PBS/MgCl2 solution (2 mM MgCl2 in PBS) for 1–2 h. Fixative was removed by multiple washes in ice cold PBS/MgCl2 and embryos were frozen on Dry Ice in OCT embedding medium. Tissue sections (25 µm) were mounted on SuperFrost/Plus slides and allowed to air dry for 1 h at 4°C. Whole mount tissues and sections were washed in ice cold PBS/MgClS to thoroughly remove OCT and then immersed in X-gal staining solution (InvivoGen, San Diego, CA; 0.02% Igepal, 0.01% sodium deoxycholate, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/ml X-gal in PBS/MgCl2) for 2–6 h in a 37°C incubator. Tissues were then washed and photographed. For immunohistochemistry following X-gal staining, sections were fixed for 1 h in ice cold 4% PFA. Slides were washed in PBS and were allowed to dry on a 37°C heater overnight. β-Gal-stained tissue was labeled with mouse anti-neurofilament antibody using the single-label immunohistochemistry protocol described above. Sections were mounted in Glycergel mounting medium (Dako North America, Carpinteria, CA, USA) and imaged using a bright field camera.
Neurons of the geniculate ganglion were quantified in transverse serial sections of the mouse embryo head. Monoclonal mouse anti-β-III tubulin antibody (TUJ-1) was used to identify and count neuron profiles, only in sections where the nucleus was visible (“*” Fig. 2J inset). Neuronal profiles were counted in six sections per geniculate ganglion. The area containing the geniculate ganglion was measured for each section and multiplied by thickness of the section (5 µm) to derive the volume. The volumes for all sections were summed to derive the volume for the entire ganglion. The total number of neuron profiles in the ganglion was estimated as the product: (number of profiles per volume of the counted section) × (total volume of the entire ganglion). The total number of neurons per ganglion was estimated by multiplying the number of total neuron profiles by a correction factor, to compensate for the presence of the nucleus in multiple sections (Abercrombie, 1946), according to the formula: N = n × [T/(T × D)] where N is the estimated total number of neurons, n is the number of nuclear profiles, T is the measured section thickness, and D is the average diameter of nuclei. This estimate was calculated separately for each ganglion based on the average diameter of 50 neuronal nuclei. Diameters were calculated from area measurements of each nucleus. This approach has been used successfully to examine changes in ganglion neuron number following manipulations of neurotrophins (Agerman et al., 2003; ElShamy and Ernfors, 1997; Erickson et al., 2001; Erickson et al., 1996).
Time-bred mouse embryos at E13.5 (wild-type, n = 3; bdnf −/−, n = 3) were euthanized and immersion fixed in 4% PFA in 0.1M PBS (pH 7.4), and transferred to 30% sucrose until the tissue sank to the bottom of the jar. Heads were then transferred to a 1:1 mixture of 30% sucrose:OCT, and allowed to equilibrate for 1–2 h. Subsequently, the heads were frozen in OCT embedding medium and stored at −80°C. Double knockouts for bdnf and the pro-apoptotic gene bax were a gift from Dr. D.M. Katz (Department of Neurosciences, Case Western Reserve University, Cleveland, OH). Embryo heads were cut in transverse serial sections (60 µm), air dried on a slide warmer for 3 h, and stained with cresyl violet. To maintain section thickness, tissues were not dehydrated and were mounted in a glycerol-based mounting medium for stereological quantification. Neurons were quantified using Stereo Investigator (version 7) software (MBF Bioscience, Williston, VT, USA). An experimenter blinded to the genotype of the animals traced a contour around the geniculate ganglion under low magnification (20×). Every section containing the ganglion was traced for a volume measurement, and cells were quantified in three of those sections. Counting parameters were optimized such that a sample of at least 300 cells was counted. Within each traced contour of the geniculate ganglion, the computer determined the placement of the counting frames randomly. The depth (z-axis) of the counting frame was equal to minimal thickness of the section, minus a total guard zone of 6 µm (3 µm from the top and bottom of the section). At 100× magnification, geniculate ganglion neurons were counted in the volume designated by each counting frame (25 µm2). Cells were counted only when they first came into focus (cell top), so that each cell was counted only once. Based on these measurements, the total volume of the ganglion was estimated using the Cavalieri method (MBF Bioscience), and the total number of cells then estimated for the entire volume of the ganglion using the optical fractionator probe (MBF Bioscience).
Geniculate ganglion cell numbers were quantified as mentioned above. Total neuron number and total volume were compared between genotypes on embryonic days E12.5, E13.5, E14.5, E16.5, and E18.5, using a two-way analysis of variance (ANOVA). The cell death data was analyzed using two way repeated measures ANOVA (one factor repetition). The Bax experiment data was analyzed using a one way ANOVA. Bonferroni’s post hoc test was used to compare individual means as needed. The significance level was set at p < 0.05 for all statistical comparisons.
To understand the timing of the geniculate ganglion neuronal reduction seen at birth in bdnf −/− mice, we quantified the number of geniculate neurons from E12.5 until E18.5 for both wild-type and bdnf −/− mice. Neuronal cells were identified using a neuronspecific anti-β-III tubulin antibody (TUJ-1), which labels only neurons and neuronal precursors (Fanarraga et al., 1999). The geniculate ganglion was easily recognizable as a distinct entity, even though it is fused with the acoustic/vestibular ganglion at ages E13.5 and earlier (Fig.1) TUJ-1-labeled cells are easily recognized by their dark stained cytoplasm and a clear nucleus (Fig. 2J insert, asterisk). Overall, there was a significant decrease in the number of neurons with embryonic age (p <0.001). In wild-type mice the number of ganglion neurons did not differ between ages E12.5, E13.5 and E14.5, but at E16.5 the neuronal number was reduced by 36% relative to E12.5 (p < 0.05, Fig. 3A). There was also a substantial decrease in neuron number in bdnf −/− mice compared with wild-type mice (p<0.001), which was age dependent. Specifically, there was no difference between wild-type and bdnf −/− embryos in the total number of geniculate ganglion neurons at E12.5. However, by embryonic day E13.5 the number of neurons in geniculate ganglia of bdnf −/− embryos was reduced by 32% relative to wild-type embryos (p < 0.01, Fig. 3A). The neuron loss at E13.5 was verified using a stereological Optical Fractionator counting method (Fig. 3B). This approach is considered more reliable than the immunohistochemical approach, in that fewer assumptions are made about the tissue. However, it also has the disadvantage in that the TUJ1 antibody for labeling neurons could not penetrate the tissue at tissue thicknesses required for stereology. Instead, neurons were identified based on morphological and nissl staining characteristics. With this method, we found a 46% loss of geniculate ganglion neurons by E13.5 in bdnf −/− mice compared to wild-type mice (p < 0.05, Fig 3B). This confirms that cells within the geniculate ganglion are lost by E13.5 of development in bdnf −/− mice. Neuron numbers continued to decrease in bdnf −/− mice, and by E18.5 there was a 65% loss in geniculate ganglion neuron number relative to wild-type littermates (p < 0.001, Fig. 3A).
Unlike neuron number, geniculate ganglion volume increased with age (p<0.003, Fig. 3C). Since neuron number does not increase, this change in volume is likely due to increases in neuron size. As with neuron number, there was a substantial reduction in the volume of the geniculate ganglion in bdnf/−/− mice compared with wild type mice (p<0.001), which was age dependent. Specifically, there were no differences between the geniculate ganglion volumes of wild-type and bdnf −/− mice at E12.5 (Fig. 3C). At E13.5 volumes were measured in both the TUJ-1 labeled paraffin sections of the ganglia and in tissue processed for stereology using the Cavalieri estimation. Although not statistically significant for paraffin sectioned ganglia (p = 0.078), geniculate ganglion volume was reduced by 42% in bdnf −/− mice compared to wild-type mice (p = 0.01, Fig. 3D) at E13.5, using the Cavalieri estimation. Because the Cavalieri estimation requires the use of tissue that is minimally dehydrated, estimated volumes are closer to actual volumes. By E14.5 the geniculate ganglion from bdnf −/− embryos was noticeably smaller (Fig. 2F), was present in fewer sections, and had a reduced volume (p < 0.05, Fig. 3C) compared to wild-type embryos. As with neuron numbers, geniculate ganglion volume continued to decrease relative to that of wild-types, such that by E18.5 geniculate ganglia from bdnf −/− embryos were 80% smaller than their wild-type counterparts (p < 0.001, Fig. 2J). By E18.5 geniculate neurons were also reduced in size by 14% in bdnf −/− embryos (118.1 ± 4.8 µm2) relative to wild-type mice (138 ± 3.9 µm2; p < 0.01). The parallel time courses for the reduction in volumes and neuron number within the geniculate ganglion indicates that BDNF is required for survival of these gustatory neurons starting early, between E12.5 and E13.5 of development but also continues to be required through later stages of development.
We found that bdnf −/− mice lose geniculate neurons over a protracted period of embryonic development, beginning before and continuing beyond the period of peak cell death (Carr et al., 2005). The loss of neurons in bdnf −/− mice could reflect an increased rate of cell death, decreased proliferation rate, or both. Functional deletion of Bax protein typically blocks apoptosis in developing sensory neurons downstream of neurotrophic factors. To determine whether geniculate ganglion neurons lacking the pro-apoptotic gene bax could survive in the absence of the neurotrophin BDNF, we compared neuron numbers using the optical dissector method for wild-type, bdnf −/−, bax−/−, and bdnf −/−/bax−/− double knockout mice at birth, after the period of developmental cell death has presumably ended. Consistent with earlier findings, geniculate neuron numbers were significantly reduced in bdnf −/− mice compared to wild-type mice (p < 0.03, Fig. 4B and Fig. 5A). In bax −/− mice, however, there was a doubling of both the neuronal number (p < 0.01, Fig. 5A) and the volume of the geniculate ganglion (p < 0.05, Fig. 4 & Fig. 5B). Thus, removal of bax enhanced the survival of the developing geniculate ganglion neurons. This finding indicates that the pro-apoptotic gene bax is required for naturally occurring developmental cell death of geniculate ganglion neurons. In contrast, the bdnf −/−/bax −/− double knockout animals had the same number of neurons as the bax−/− mice within the geniculate ganglion (Fig. 5B). Thus, removal of bax completely eliminated the cell loss due to the removal of BDNF. These findings indicate that BDNF normally functions to prevent cell death via the Bax-dependent cell death pathway.
A developing ganglion consists of an undifferentiated precursor pool, a transient amplifying population of neuronal precursors, mature neurons, and glia. Neurotrophins have been shown to influence the survival of both neuronal precursors and differentiated neurons (Ernfors, 2001; Farinas et al., 2002; Liebl et al., 2000; Lopez-Sanchez and Frade, 2002). Here, we found that the loss of geniculate ganglion neurons begins at E13.5 in bdnf −/− mice. Thus, we selected this age to determine which cells are dying within the geniculate ganglion. Cells undergoing cell death were identified using an antibody against activated caspase-3 (Fig. 6A,D). Caspase-3 is a member of the pro-apoptotic caspase family that is synthesized as an inactive pro-enzyme and processed to active form in cells undergoing apoptosis. Thus, caspase-3 acts as a relevant marker for developmental cell death (De Zio et al., 2005). We quantified the number of precursors using BrdU, a cell proliferation marker (Fig. 6B,E). Differentiated neurons were enumerated using an antibody against neurofilament (Fig. 6C,F), an intermediate filament found specifically in neurons (Debus et al., 1982; Shaw et al., 1984). Differentiated neurons undergoing cell death were labeled by antibodies to both activated caspase-3 and neurofilament (Fig. 6G,H,I). In the absence of BDNF, a 65% increase in geniculate ganglion cells positively labeled for activated caspase-3 was observed (p < 0.01, Fig. 7). These observations are consistent with an increase in activated caspase-3-dependent cell death in bdnf −/− mice at E13.5. No significant difference was seen for the number of BrdU positive cells in bdnf −/− vs wild-type embryos (Fig. 7). Also, no cells were co-labeled for neurofilament and BrdU in either wild type or bdnf −/− mice, this finding indicates that neurons that express neurofilament have exited the cell cycle phase of development. In addition, we found no neurons that were co-labeled with either caspase-3 or BrdU, indicating that proliferating cells are not dying in either wild type or bdnf −/− mice. This implies that BDNF does not influence the dividing geniculate cell population during development. On the other hand, the number of double-labeled cells for activated caspase-3 and neurofilament increased by 6-fold in bdnf −/− as compared to wild-type ganglia cells (p < 0.01, Fig. 7). This finding indicates that BDNF is required for survival of differentiated geniculate ganglion neurons. We did not observe cells labeled for all the three antigens.
Geniculate ganglion neuron loss begins at E13.5, suggesting that the presence of BDNF in the gustatory system is essential beginning at this early embryonic age. Several sources may provide the BDNF necessary for survival of geniculate neurons at E13.5. Neurotrophins are frequently produced by peripheral targets in minimal amounts to enable survival of correctly wired neurons (Huang and Reichardt, 2001; Vogel and Davies, 1991). However, neurotrophins are also produced within sensory ganglia as well as along projection pathways (Farinas et al., 1996). To identify the source of BDNF at E13.5, we used genetically engineered mice with a lacZ reporter gene inserted in place of BDNF in one allele (Yee et al., 2003). Thus, the pattern of β-Gal acted as a visible reporter for endogenous BDNF expression. The geniculate ganglion showed only tiny spots of β-gal reaction product (Fig. 8B), indicating that overall BDNF expression was not robust (Fig. 8A).
Geniculate ganglion neurons innervate three targets: taste buds on the palate (soft palate and nasoincisor ducts), taste buds of the tongue, and skin of the external ear. The developing palatal shelves did not show any apparent β-Gal staining at E13.5 within either whole mount (Fig. 8C) or coronal sections (Fig. 8D). Thus, the developing soft palate is not a primary source of BDNF for developing geniculate neurons at E13.5. In the lingual epithelium, the developing fungiform placodes, where taste buds will eventually develop, showed intense β-Gal staining, demonstrating robust BDNF expression, specifically in the fungiform placodes (Fig. 9C). Note that the epithelium surrounding the fungiform placodes is completely devoid of any β-Gal staining. Furthermore, the intense β-Gal staining present in the placode appears to be limited to the epithelium (Fig. 9C, arrowheads indicated epithelial mesenchyme border). In the sagittal sections of the tongue, diffuse β-Gal staining was visible in the base, where the nerve enters (Fig. 9D,F). The mesenchyme surrounding the peripheral sensory fiber bundles also showed diffuse β-Gal spots, seen clearly in blue (Fig. 9E). BDNF expression within the tongue epithelium is limited to the gustatory targets. Although the gustatory fibers have not invaded the epithelium at E13.5 (Fig. 9G,H), the fibers have just reached the fungiform placodes and are in a position to detect the BDNF produced there. The external ear also showed substantial amounts of β-gal staining (Fig. 9I,J), indicating that this target also expresses BDNF.
The central branches of geniculate neurons project to the nucleus of the solitary tract in the brainstem. We did not observe β-Gal staining in the brainstem at E13.5 (data not shown). Of the tissues examined, the two most robust sources of BDNF were the fungiform papillae and the external ear. While this does not exclude BDNF from other sources, our results indicate that the primary sources of BDNF at E13.5, when geniculate neurons are first BDNF-dependent, are the peripheral targets of these neurons.
Peripheral developing ganglia are formed by the migration of neural crest-derived or placodal-derived progenitor cells that undergo several cell cycles, resulting in an over production of neurons, which are then reduced in number via cell death. Neurotrophins could potentially influence total neuron number by regulating various stages of this process. The timing of neuron loss in neurotrophin null mice indicates a great deal about their function. For example, neurons in the DRG of nt-3 −/− mice are first lost between E11–E13, which is well before target innervation (Farinas et al., 1996). Alternatively, in ngf −/− mice, DRG neurons are first lost between E13.5 – E15.5 (White et al., 1996). Therefore, NGF may function purely as a target-derived neurotrophic factor in this system, while NT3 cannot. BDNF is required to maintain embryonic geniculate neuron number is in vivo and in vitro (Al-Hadlaq et al., 2003; Conover et al., 1995). Here, we demonstrate that in the absence of BDNF, the geniculate ganglion initially loses 46% of its cells by E13.5. This loss occurs just as fungiform placodes/papillae are starting to become innervated. The tongue fungiform papillae, which eventually house taste buds, become innervated between E13.5 and E14.5, and directed growth by the chorda tympani toward these papillae has been observed by E13.5 (Mbiene, 2004). Consistent with these studies, we also observed that many chorda tympani fibers end just below the epithelial surface by E13.5. In addition, removal of BDNF from the epithelium on or before E13.5 disrupts target innervation, while removal of BDNF after E14.5 does not (Ma et al., 2009). Taken together these findings indicate that geniculate neurons are first lost in bdnf−/− mice as chorda tympani axons become directed toward and are about to innervate their targets. This is likely the earliest age at which BDNF expressed by the gustatory placodes could influence chorda tympani neuron development.
While the timing of neuron loss is consistent with a target-derived neurotrophic factor role for BDNF in the taste system, it occurs much earlier than might be expected based on studies of normally occurring cell death in this system. In rat, naturally occurring cell death peaks at later embryonic stages (Carr et al., 2005) and occurs concurrently with a decrease in BDNF expression within the fungiform papillae (Nosrat and Olson, 1995). We observed a loss of geniculate neurons in wild-type mice by E16.5, consistent with a peak cell death between E14.5 and E16.5 in the mouse. Therefore, geniculate neurons become BDNF-dependent as they innervate their targets, but before naturally occurring developmental cell death. BDNF also regulates neuron loss in the geniculate ganglion earlier than other BDNF-dependent sensory systems. The initial vestibular and visceral BDNF-dependent sensory neuron loss occurs well after, rather than concurrent with, target innervation in bdnf −/− mice (Brady et al., 1999; ElShamy and Ernfors, 1997; Erickson et al., 2001; Ernfors, 2001). Therefore, the timing between onset of BDNF dependence and target innervation seems to vary among different BDNF-dependent sensory neuron populations.
Although the first major loss of geniculate ganglion neurons is observed at E13.5 in BDNF-null mice, this loss continues to increase until E18.5, when 65% fewer neurons exist in bdnf −/− mice than in wild-type littermates. Thus, neuron loss continues at a greater rate in bdnf −/− mice than in wild-type mice throughout the normal period of geniculate ganglion cell death (Carr et al., 2005). The soft palate is innervated 1 day later than the tongue, at E15.5 (Nakayama et al., 2008). Therefore, neurons innervating the soft palate may be initially lost after E13.5, contributing to the later neuronal losses. In addition, the continued loss of neurons in bdnf −/− ganglia is consistent with late postnatal loss of chorda tympani fibers from the tongue. In the BDNF-null animals, targeting is disrupted, and chorda tympani fibers branch extensively near the epithelial surface (Ma et al., in press). By E18.5, some of these fibers innervate the correct target, but most withdraw. This loss of peripheral innervation occurs between E16.5 and E18.5, corresponding to the later neuronal losses observed in bdnf −/− mice. Thus, the continued loss of geniculate ganglion neurons after E13.5 in the absence of BDNF could be explained, in part, by the difference in timing of innervation to the tongue and soft palate. In addition, this continued neuron loss explains the relatively late withdrawal of afferent fibers innervating the tongue in bdnf −/− mice.
Developmental neuronal death has been attributed to a Bax-dependent programmed cell death pathway (White et al., 1998). Bax is a member of the Bcl-2 family of proteins that play key roles in programmed cell death via the intrinsic or mitochondrial pathway. We found that normal developmental cell death in the geniculate ganglion requires Bax. Furthermore, we demonstrated that Bax removal rescues neuron losses in BDNF-null mice demonstrating that BDNF can only regulate geniculate neuron number when Bax is also present. Thus, BDNF enables survival in this ganglion by suppressing the Bax-dependent cell death pathway. This finding is consistent with previous demonstrations that Bax-null mutation rescues BDNF-dependent neuronal loss in the developing nodosepetrosal ganglion as well as in the visceral ganglion (Hellard et al., 2004). Other neurotrophins have also been shown to inhibit the Bax-dependent cell death pathway to allow neuron survival. NGF- and NT-3-dependant spinal sensory neuron loss can be rescued via inhibition of the Bax-dependent cell death pathway (Patel et al., 2000; Patel et al., 2003). Thus, inhibition of the Bax-dependent cell death pathway is a common mechanism employed by neurotrophins to support survival of developing sensory neuronal populations.
While both Bax inhibition and caspase-3 activation are common mechanisms of neurotrophin action, neither are universal. Death in different neuronal types might result from the activation of varying arrays of caspases, activation of these molecules commonly results in caspase-3 activation for many neurons (Yuan and Yankner, 2000). Caspase-3 typically functions downstream of Bax and cleaves different substrates to facilitate cell death via DNA fragmentation (De Zio et al., 2005; Earnshaw et al., 1999). Geniculate ganglia from bdnf −/− mice had a higher number of neurons expressing activated caspase-3 than wild type mice. Thus, BDNF prevents neuron death through a caspase-3 dependent mechanism. Interestingly, caspase-3 is also activated in some wild-type neurons, indicating that normal developmental cell death is mediated by the same mechanisms as BDNF deficiencies.
The stage of development at which neurons die in neurotrophin mutants varies across sensory neuron populations and neurotrophins. We found that proliferating cells (precursors) in the geniculate ganglion were unaffected by BDNF removal and that no precursor cells also contained activated caspase 3 in either wild type or bdnf −/− mice. This is very different from the DRG, where NT-3 has been shown to regulate the precursor population (Elshamy and Ernfors, 1996; Farinas et al., 2002; Farinas et al., 1996). BDNF removal increased activated caspase 3 specifically with in cells that also expressed neurofilament. Neurofilaments are typically expressed in differentiated neurons, but can also be expressed in late stage neuronal precursors (Hall and Landis, 1991; Tapscott et al., 1981). However, we found no cells positively labeled with neurofilament and BrdU, indicating that neurofilament positive precursors were either non-existent or so few as to remain undetected. It is possible that the presence or absence of neurofilament positive neuronal precursors depends on neuron location and age. Regardless, we did not see any neurofilament positive cells that were also proliferating in the geniculate ganglion at E13.5. Thus, it appears that it is largely differentiated neurons undergoing increased cell death in the geniculate ganglion in response to BDNF removal. BDNF may function similarly in other BDNF-dependent sensory populations (Brady et al., 1999; ElShamy and Ernfors, 1997; Erickson et al., 2001; Ernfors, 2001); however, geniculate neurons are the first BDNF-dependent sensory population in which neuron proliferation and death have been specifically examined.
Because differentiated geniculate neurons require BDNF for survival beginning at E13.5, we examined the possible sources of BDNF available to geniculate neurons at this age. It is well established that BDNF is expressed in developing fungiform papillae and in the soft palate during embryonic rat development (Nosrat, 1998; Nosrat et al., 1996; Nosrat and Olson, 1995), and also in developing rat geniculate ganglia (Schecterson and Bothwell, 1992). Similar to rat (Nosrat and Olson, 1995), we show that at E13.5 the developing fungiform papillae in the mouse tongue are a major source of BDNF. Another target of geniculate neurons, the external ear, also appears to be a primary source of BDNF for developing geniculate neurons at E13.5. Interestingly, BDNF expression is not robust in the soft palate at E13.5. This subpopulation of geniculate neurons may not be dependent on BDNF. Roughly, one-third of the geniculate neurons innervate each of the three major targets of the geniculate ganglion (Gomez, 1978; Semba et al., 1984), therefore, it is certainly possible that the missing neurons only innervate tongue and the external ear. More likely, BDNF expression may be initiated and may support neuron survival at later stages of development than for the front two-thirds of the tongue, because target innervation occurs later in the soft palate than in the tongue. The base of the tongue and the geniculate ganglion produced relatively small amounts of BDNF. In addition to being sparse, the βgal-reaction is very punctate which could represent the degradation of the beta-gal protein in contrast to the cytoplasmic labeling characteristic of cells actively synthesizing beta-galactosidase. This would imply that while these cells did synthesize beta-gal and therefore BDNF at previous stages of development, they are no longer actively producing protein at E13.5. We did not observe BDNF-lacZ expression in the hindbrain at E13.5. Therefore, BDNF in these locations is less likely to contribute to geniculate neuron survival at E13.5 than BDNF in the target. This hypothesis is consistent with our observations that neurons survive independently of BDNF until their axons near their peripheral targets, indicating that BDNF produced along the path of innervation is not essential for survival of these neurons. Thus, the robust expression of BDNF in the developing fungiform papillae and outer ear at the time point of initial neuron loss indicates that BDNF produced by the terminal targets is essential for survival of these neurons.
Our findings indicate that during normal development, geniculate ganglion neurons differentiate and extend axons to innervate the tongue independently of BDNF. By E13.5, these axons approach the developing fungiform papillae and the developing ear, where robust levels of BDNF are produced. At this time, developing geniculate neurons become dependent on BDNF for survival. BDNF expressed in fungiform papillae or in the external ear suppresses the death of differentiated geniculate ganglion neurons by inhibiting the activation of Bax and caspase-3. At later embryonic ages, BDNF expression in the fungiform papillae decreases (Nosrat and Olson, 1995), resulting in neuronal death by activation of both Bax and caspase-3.
David M. Katz for providing the hybrid bdnf−/−/bax−/− animals. This work was supported by National Institutes of Health Grant DC007176 (RFK).
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