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Dissection of NT3 functions in vivo by gene replacement strategy

¹ Neural Development Group, Mouse Cancer Genetics Program, NCI, Frederick, MD 21701, USA
² Department of Neurology, Boston University School of Medicine, Boston, MA 02118, USA
³ Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA

View larger version (48K): [in a new window]   Fig. 1. Replacement of the endogenous NT3-coding region with BDNF. (A) Diagram of the replacement vector and strategy used to ‘knock-in’ BDNF into the NT3 locus. Probes external to the targeting vector were the same as previously described (Tessarollo et al., 1994). Successful targeting of the locus with the replacement vector is revealed by a change of the wild-type-specific 6.5 kb ScaI DNA restriction fragment (wt) to an 8.5 kb ScaI fragment (mt). Removal of the neo selectable marker cassette by CRE-mediated recombination of the targeted allele is indicated by the switch of the 8.5 kb ScaI fragment (mt) to a 7.0 kb fragment (mt (cre)). X, XbaI; S, ScaI; B, BamHI; E, EcoRI; Sm, SmaI. (B) Southern blot analysis of DNA from wild-type embryonic stem (ES) cells (+/+), a heterozygous B/N ES cell clone (+/mt), and a heterozygous B/N ES cell clone following Cre-mediated recombination (+/mt (cre)). The ScaI restriction enzyme digestion and the (NT3) 5' probe indicated in A were used to detect the rearrangements in the murine NT3 locus. The BDNF-coding region (BDNF) and the neo (neo) probes were hybridized to parallel blots. Bacteriophage lambda DNA cut with HindIII was used as the size markers. (C) To ensure that the recombinant vector could produce biologically active BDNF, we introduced the NT3/BDNF chimeric genomic sequence into the pMEX vector which drives expression from an MSV-LTR promoter (Martin-Zanca et al., 1989). The supernatant of NIH 3T3 cells transfected with the pMEXneo-BDNF expression vector was harvested and tested on TrkB-expressing PC12 cells for the presence of BDNF activity. The presence of BDNF in the media was revealed by neurite outgrowth (bottom right). Unconditioned media (top left) and conditioned media by NIH 3T3 cells transfected with the pMEXneo (top right) did not induce neurite extension. Treatment of the PC12 cells with 1ng/ml recombinant BDNF was used as positive control (bottom left).

View larger version (71K): [in a new window]   Fig. 2. BDNF expression in B/N mutant mice. (A-C) Northern blot analysis of BDNF transcripts in tissues dissected from newborn wild-type (WT) and B/N mice. For each sample, 10 µg of total RNA were analyzed. The NT3-coding sequence used to probe the wild-type samples identified a unique 1.4 Kb band (A). Hybridization of the B/N tissues with the NT3 probe resulted in the total absence of signal, as expected (not shown). Hybridization of the WT samples with the BDNF cDNA probe identified the expected 1.6 and 4.0 kb bands corresponding to the endogenous products of the Bdnf gene (B) (Maisonpierre et al., 1990). Hybridization of the B/N tissues with the BDNF probe revealed, in addition to the endogenous BDNF-specific pattern of expression, an additional band (B/N) of approximately 1.4 kb (C). Note that the pattern of expression of the recombinant BDNF (B/N) parallels that of NT3. Blots were hybridized with a cyclophilin-specific probe to assure equal RNA loading (not shown). B.A.T., brown adipose tissue. (D-K) BDNF replaces NT3 expression in the mutant B/N mouse at the cellular level. In situ hybridization analysis of NT3 and BDNF expression in wild-type and B/N animals during embryonic development. Transverse dark-field views of wild-type (D-G) and B/N (H-K) embryos at embryonic stage E11.5 (D,F,H,J) and E13.5 (E,G,I,K) hybridized with an NT3- (D,E,H,I) or BDNF-specific (F,G,J,K) probe. Note the precise replacement of NT3 specific signal in the lateral motor column (arrows) of the wild-type spinal cord (D,E) by BDNF hybridization in the B/N mutant. (J,K).

View larger version (78K): [in a new window]   Fig. 3. Development of central fibers in B/N mutant mice. Retrograde labeling of sensory neurons and motoneurons was performed using the lipid-soluble fluorescent tracer DiI in axial muscles (A-C,G) or dorsal root ganglia (D-F,H) at the lumbar level (see Materials and Methods). Micrographs of sections, through the lumbar spinal cord of E 15.5 embryos after DiI tracing show a normal complement of Ia fibers in control mice (arrowheads, A,D), which is absent in Nt3-/- mutant mice (B,E). Sections through the spinal cord of a B/N mouse reveals the presence of a some fibers projecting ventrally in the spinal cord (arrowheads, C,F). Labeling performed in the axial muscles (G) or in the DRG (H) of B/N mice at stage P0 shows the presence of only residual central fibers (arrows). Immunohistochemical analysis of P0 spinal cords from wild-type (I), Nt3-/- (J) or B/N (K) animals using an antibody against parvalbumin (PV), a marker specific for proprioception fibers, shows presence of PV-specific staining in only the wild-type spinal cord (arrows, I).

View larger version (117K): [in a new window]   Fig. 4. Expression of Trk receptors in DRG neurons of B/N mutant embryos. In situ hybridization analysis of TrkA (A-F), TrkB (G-L) and TrkC (M-R) expression in transverse sections of wild-type (WT) and mutant (B/N) animals between day 11.5 and day 13.5 of embryonic development. In the spinal cord, the expression patterns of Trk genes are similar in both wild-type and B/N embryos suggesting that neurotrophins do not influence the expression of their receptor in these neurons. In the DRG of B/N mutant mice, there is a reduction in TrkC expression already at E11.5 (P) and by E 12.5 (Q) there are no TrkC-expressing neurons. TrkB-positive neurons, however, are present in the B/N embryos at all stages (J-L) in a similar fashion as observed in the wild-type DRG (G-I).

View larger version (21K): [in a new window]   Fig. 5. DRG neuron numbers in NT3- or TrkC-deficient and B/N mutant mice. Graphic representation of the number of neurons in cervical 1 (C1), thoracic 1 (T1) and lumbar 1 (L1) DRG at E12.5, E13.5, E14.5 and P0 as shown in Table 1. Data from mutant mice are expressed as a percentage of the neuron numbers of wild-type littermates. * indicates significant differences (P<0.05) between strains.

View larger version (138K): [in a new window]   Fig. 6. Rescue of cochlear innervation by the B/N allele at P1. Whole mount of transganglionically retrograde labeled ears showing the distribution of afferent fibers to the basal turn in a Nt3-/- mutant (A,D), a wild-type control (B,G) and a B/N mutant mouse at P1 (C,E,F). The B/N mouse shows a dense complement of spiral ganglion neurons and radial fibers throughout the basal turn up to the basal end of the cochlea (Base) similar to the wild-type mouse. In contrast to an almost complete absence of basal turn radial fibers and reduced density of the outer hair cell innervation in the Nt3-/- mice (D), the B/N mutant ear shows dense radial fibers (E) and innervation of both outer hair (OHC) and inner hair cells (IHC). Note that the density of the outer hair cell innervation appears to be even higher in the B/N mice than in wild-type control animals when similar areas of the cochlea are compared (B,C,F,G). Scale bars: 100 µm.

View larger version (126K): [in a new window]   Fig. 7. Basal turn cochlear innervation at P7 in wild-type and B/N mice. A collapsed z-series of confocal images shows the radial fibers (RF) extending to the inner hair cells (IHC) and outer hair cells (OHC) in the control animal (A). The innervation of the outer hair cells is less well organized in the organ of Corti in mutant animals (C) and additional fibers can be seen running underneath the organ of Corti in a separate bundle (* in B) and between the radial fibers (^ in B). None of these fibers exists in control animals. Radial sections reveal the presence of longitudinally cut radial fibers in the control animals (D) and additional transversally cut spiraling fiber bundles (^,*) below the organ of Corti among the tympanic border cells in the mutant mice (E). These fibers may be myelinated (^ in E). Immunohistochemistry (red) for acetylated tubulin (F,G) can visualize these fibers only in the mutant (^,* in G). Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 8. Schematic of NT3 activation of Trk receptors during DRG development.

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