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doi: 10.1242/10.1242/dev.00378

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BDNF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development

¹ Unit of Molecular Neurobiology, MBB, Karolinska Institutet, 171 77 Stockholm, Sweden
² Inserm U432, CNRS, Université de Montpellier 2, Montpellier, France
³ Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
⁴ Laboratory of Oral Neurobiology, Department Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI, USA

View larger version (32K): [in a new window]   Fig. 1. Gene replacement of the BDNF coding sequence with NT3. (A) Schematic diagram of the approach to change the BDNF loci to NT3. The BDNF exon V (shaded box) was opened with BglII (B) and the NT3 coding region was fused in-frame followed by a neomycin cassette. (B) ES cells were analysed for homologous recombination using an external probe for Southern blot analysis (Bs; BstXI and E; EcoRV). The wild-type (WT) allele revealed a 12 kb band while the mutant band was 5.6 kb. (C) Animals were mated with deleter-cre mice to lox out the neomycin cassette. (D-E) Two PCRs were performed to distinguish between animals containing or not containing the neomycin cassette. The PCR primers used for this analysis are depicted in (C). (D) The first PCR primers (Neo-3', Neo-5') are designed to amplify a fragment within the neomycin cassette of about 600 bp. The neomycin fragment is amplified in BDNFNT3/NT3 mice while no fragment is amplified in mice crossed with deleter-cre mice (BDNFNT3lox/NT3lox mice). (E) In the BDNFNT3lox/NT3lox mice a fragment of about 600 bp is instead amplified using the NT3-04 and BL2 primers, this band is not amplified in animals still carrying the neomycin cassette.

View larger version (58K): [in a new window]   Fig. 2. mRNA and protein expression in the BDNFNT3/NT3 mice at P14. (A-D) BDNF and NT3 in situ hybridisation of brain sections. Scale bars:100 µm in A-D. Note that NT3 mRNA expression changes in BDNFNT3/+ (C) and BDNFNT3/NT3 (D) mice towards the same expression pattern as BDNF in wild-type animals (A). (E-F) Analyses of protein levels in hippocampus using BDNF and NT3 ELISA. (E) BDNF protein levels decrease in BDNFNT3/+ mice and are completely absent in BDNFNT3/NT3 animals. (F) NT3 protein levels increase in BDNFNT3/+ and BDNFNT3/NT3 mice to reach a similar level as BDNF protein in wild-type animals.

View larger version (49K): [in a new window]   Fig. 3. Rescue of neuronal numbers, target innervation and function in the cochlea in BDNFNT3/NT3 mice. (A) BDNF and NT3 expression in the cochlea during development and in adult animals. BDNF (red) is expressed in the hair cells during development while NT3 (blue) is expressed in both hair cells and supporting cells. In adult animals NT3 is only expressed in the IHC. (B) Auditory brainstem response in BDNF–/– (green), BDNFNT3/NT3 (red) and wild-type (blue) mice. Note that the BDNF–/– mice suffer from a hearing loss that is completely rescued in the BDNFNT3/NT3 mice. (C,D) Afferent innervation in sections from the apical to middle part of the cochlea stained with an antibody directed against p75NTR (red) counterstained with phalloidin (green) to visualise the hair cells. Scale bars: 20 µm. The arrows indicate the OHC. Note innervation of the three rows of OHCs in the BDNFNT3/NT3 mice. (E) Spiral ganglion neurons (SGN) were counted at P0 and P17. At P0 25% (n=3) and at P17 29% (n=4) of the neurons were lost in BDNF–/– mice compared to wild-type mice. This loss was rescued at P0 in BDNFNT3/NT3 mice (n=3) and at P17 there was still no significant difference between the wild-type (n=4) and BDNFNT3/NT3 mice (n=3). ANOVA Fischer's ***P<0.001; ±s.e.m.

View larger version (111K): [in a new window]   Fig. 4. DiI tracing of afferent innervation in wild type (A,D,G,J) BDNF–/– (B,E,H,K) and BDNFNT3/NT3 (C,F,I,L), and whole mount immunohistochemistry to detect radial fibres in wild type (M) BDNF–/– (N) and BDNFNT3/NT3 (O). Micrographs illustrate the apical (A-F,M-O), middle (G-I), and base (J-L) of the cochlea. Scale bars: 40 µm in A-C and M-O and 10 µm in D-L. There is no reduction in the density of radial fibres (rf) in either BDNF–/– (B) or BDNFNT3/NT3 (C) compared to wild-type (A) mice as seen by DiI tracing. This is also shown by the distribution of nerve fibres stained for acetylated tubulin (red) and counterstained with FITC-conjugated phalloidin (green) to visualise the hair cells (M-O). Note the innervation of the three rows of OHCs in wild-type mice (D,G,J), loss of innervation of all three rows of OHCs in BDNF–/– mice (E,H,K) and rescue of innervation of the OHCs in the BDNFNT3/NT3 mice (F,I,L). Arrows indicate the three rows of OHC.

View larger version (64K): [in a new window]   Fig. 5. Neuronal survival but not innervation is rescued in the vestibular system. (A) BDNF and NT3 expression in the vestibule during development and in adult animals. Both BDNF (red) and NT3 (blue) are expressed in the sensory epithelia during development, while only BDNF is expressed in the adult. (B) In P0 BDNFNT3/NT3 mice, 60% of the neurons survived compared to only 16% survival in BDNF–/– mice. At P17 half of the neurons seen at P0 were lost in BDNFNT3/NT3 mice. (C-D) Sections of utricular maculae immunohistochemically stained with an antibody directed against p75NTR (red) that specifically stains the afferents and with phalloidin (green) to visualise the hair cells. Scale bars: 25 µm in C and D. Only a sparse innervation that does not form calyces is seen in the BDNFNT3/NT3 mice (D) compared to wild-type animals (C). ANOVA Fischer's ***P<0.001; ±s.e.m.

View larger version (95K): [in a new window]   Fig. 6. Failure of terminal nerve in-growth in BDNF–/– and BDNFNT3/NT3 mice. (A-I) Immunohistochemistry for p75 to detect afferent innervation (red) and FITC-conjugated phalloidin (green), which visualises the hair cells. (A-C) Already at E16 when the fibres have reached the epithelium of the utricular and saccular maculae in wild-type mice (A), BDNFNT3/NT3 mice showed a comparable failure of innervation (C) to BDNF–/– mice (B). (D-F) Similar results were obtained at P0. Note that only a few fibres have reached the hair cells in the BDNFNT3/NT3 mice (F) and none has reached the hair cells in BDNF–/– mice (E). Nerve fibres are seen at all stages in the subepithelial layer, suggesting that BDNF is only required for terminal innervation and formation of functional nerve endings but that it is not required for nerve fibres to project to the utricular and saccular compartments. (G-I) The ampullar cristae completely lack innervation in both BDNF–/– (H) and BDNFNT3/NT3 mice (I). Scale bars: 50 µm in A-I.

View larger version (70K): [in a new window]   Fig. 7. BDNF/TrkB signalling is required for terminal innervation and formation of functional sensory nerve endings. (A-H) Immunohistochemical double staining against NF200 to detect nerve fibres (red) and calretinin to detect calyces (green). (A,B) Whole-mount preparations from the utricular maculae in wild-type (A) and BDNFNT3/NT3 mice (B). Scale bars: 30 µm in A and B. The analyses using confocal microscopy confirmed a sparse innervation of the epithelia and also revealed that the nerve fibres in the subepithelial layer were highly disorganised in BDNFNT3/NT3 mice (B). (C-H) Co-cultures of wild-type vestibular ganglion neurons with wild-type hair cells (C-D) BDNF–/– hair cells (E-F), or BDNFNT3/NT3 hair cells (G-H). Scale bars: 20 µm in C-H. With wild-type hair cells the nerve endings branch extensively and wrap around the hair cells forming digitations similar of the process of calyx formation in vivo (C, nerve including calretinin-stained hair cells and D, nerve only). In only two out of 200 cases did wild-type neurons co-cultured with BDNF–/– hair cells contact a hair cell (E-F). Note that the diameter of the nerve fibre is much thinner and it shows less branching on the surface of the BDNF–/– hair cell. (G,H) In contrast to BDNF–/– hair cells, nearly all BDNFNT3/NT3 hair cell fragments were contacted by vestibular neurons (11 out of 12). However the contacting neurites remained undifferentiated on the surface of the epithelia and did not develop digitations.

View larger version (120K): [in a new window]   Fig. 8. NT3 cannot support neuronal survival or innervation in the tongue. (A) Schematic drawing of BDNF (red) and NT3 (light blue) mRNA expression in fungiform papilla in rodents. The arrow indicates the taste bud with innervation. The yellow lines represent the BDNF-dependent intragemmal fibres (gustatory fibres) and the dark blue dashed lines represent perigemmal fibres (somatosensory innervation). Gustatory nerve fibres innervate only the taste buds where BDNF is present while somatosensory nerve fibres predominantly innervate the surrounding epithelium where NT3 is present. (B) Typical innervation of a fungiform papilla and its taste bud (arrow) in a wild-type P7 mouse tongue, visualised by immunohistochemistry against protein gene product 9.5 (PGP). The papilla is well-innervated, gustatory fibres innervate the taste bud (arrow) and perigemmal somatosensory nerve fibres innervate the surrounding epithelium. (C) An example of fungiform papilla innervation in BDNFNT3/NT3 mice. The NT3-dependent somatosensory innervation apparatus of the papilla appears intact, while the taste bud innervation (arrow) is almost non-existent. (D) Another example of fungiform papilla innervation in BDNFNT3/NT3 mice. Generally, the papilla is malformed. It has filiform papilla morphology but could be distinguished from a normal filiform papilla by the amount of nerve fibres in this papilla. An under-developed taste bud seems to be present in the core part of the papilla (arrow). (E-G) Scanning electron micrographs of the anterior part of the tongue from P7 mice. (E) Fungiform papillae rise above the filiform papillae in a wild-type tongue. (F) Surface morphology of the BDNFNT3/NT3 tongue reveals major loss of fungiform papillae and the few remaining papillae are reduced in size. (G) Anterior tongue morphology in BDNF–/– mice is similar to that of the BDNFNT3/NT3 mice. (H) Quantitative measurements of P7 geniculate ganglion neuronal numbers. Half of the neurons are missing in the BDNF–/– and BDNFNT3/NT3 mice compared with wild-type animals. The neuronal numbers show no significant difference between BDNF–/– and BDNFNT3/NT3 mice. ANOVA Fischer's ***P<0.001; ±s.e.m. Scale bars: 100 µm in B-D and 200 µm in E-G.