Loss of brain-derived neurotrophic factor-dependent neural crest-derived sensory neurons in neurotrophin-4 mutant mice - PubMed (original) (raw)

Loss of brain-derived neurotrophic factor-dependent neural crest-derived sensory neurons in neurotrophin-4 mutant mice

D J Liebl et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Peripheral ganglion neurons confer sensory information including touch, pain, temperature, and proprioception. Sensory modality is linked to specific neurotrophin (NTF) requirements. NT-3 supports survival of neurons that differentiate primarily into proprioceptors whereas nerve growth factor and brain-derived neurotrophic factor (BDNF) support subpopulations that transmit nociception and mechanoreception, respectively. We examined sensory neurons of gene-targeted mouse mutants at the NT-4, BDNF, NT-3, and TrkA loci. We show that NT-4 functions early in gangliogenesis, upstream of BDNF. In the absence of NT-4 function, BDNF-dependent, TrkB-expressing neurons fail to appear. The results are consistent with the model that precursor cells intended to become BDNF-dependent mechanoreceptors instead differentiate into NT-3-dependent proprioceptive neurons.

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Figures

Figure 1

Figure 1

Generation of NT-4 and TrkA mutant mice. (a) Schematic diagram showing the replacement vector and strategy used to inactivate the NT-4 locus. The NT-4 coding region is indicated. Restriction enzyme sites are as indicated: Sc, _Sca_I; RI, _Eco_RI; St, _Stu_I; and RV, _Eco_RV. (b) Southern blot analysis of tail DNA from a litter obtained by intercrossing two NT-4 +/− mice. _Sca_I restriction enzyme digestion and the 3′ probe indicated in a were employed to detect rearrangement in the mouse NT-4 locus. The 5.5-kb wild-type (WT) and 18-kb rearranged (mt) DNA bands are indicated. (c) Schematic diagram showing the replacement vector and strategy used to inactivate the TrkA locus. Solid boxes indicate the TrkA exons containing the translation start site (ATG) and the extracellular domain exon (ED) corresponding to nucleotide sequence 918-1247 of the TrkA cDNA. Restriction enzyme sites are as indicated: B, _Bam_HI; RI, _Eco_RI; X, _Xho_I; and H, _Hin_dIII. pBS indicates the pBluescript cloning vector (Stratagene). (d) Southern blot analysis of tail DNA from a litter obtained by intercrossing two TrkA +/− mice. _Bam_HI restriction enzyme digestion and the 3′ probe indicated in a were used to detect rearrangements in the mouse TrkA locus. The 13-kb wild-type (WT) and 11-kb rearranged (mt) DNA fragments are indicated.

Figure 2

Figure 2

Neurotrophin requirements in developing DRG. (a) TUNEL staining was examined at E11 in wt, BDNF, NT-4, and BDNF/NT-4 mutant mice. (b) Peripherin-immunostained cells in wt and mutant DRG. (_c_– e) Primary culture of wt, BDNF, NT-4, and BDNF/NT-4 mutant E13 DRG neurons incubated with 2 ng/ml BDNF (c), 2 ng/ml NT-4 (d), and 10 pg/ml NT-3 (e). (f) Primary DRG neurons cultured in the presence of NGF, BDNF, and NT-3. Small-soma-diameter neurons (arrowhead) are NGF-dependent, whereas large-soma-diameter neurons (arrow) are BDNF- or NT-3-dependent.

Figure 3

Figure 3

Anti-TrkB antibody staining (arrows) of neonatal DRG in wt (a and b) and NT-4 mutant (c and d) mice. Dark-field view of TrkC mRNA expression in neurons (arrows) of neonatal DRG in wt (e and f) and NT-4 mutant (g and h) mice. (i and j) Higher, bright-field magnification of g and h. (i and j) Arrows indicate “positive,” TrkC-expressing neurons whereas arrowheads indicate “negative,” TrkC-expressing neurons. Digoxigenin-labeled NT-4-specific antisense and sense probes (_k_– o) were hybridized to E10 embryo sections of P0, wt, and NT-4 mutant DRG. (k and l) Transverse thoracic section of E10 embryo showing hybridized (Left) and Nissl-stained tissue (Right). Arrows point to region of forming DRG. NT-4 mRNA expression in neonatal wt (m and o) and NT-4 mutant (n) DRG using antisense (m and n) and sense (o) probes. (p) DRG sections from neonatal wt, NT-4, and BDNF mutant mice were immunostained with antibodies for somatostatin (SS), substance P (SP), calcitonin gene-related protein (cGRP), parvalbumin (PA), and SSEA-3. (q) Number of myelinated axons innervating cutaneous tissue (saphenous nerve) and muscle (common peroneal and tibial nerves) in wt and NT-4 mutant mice. (r) Coordination of wt and NT-4 mutant on a rotating rod over 4 consecutive days.

Figure 4

Figure 4

Schematic diagram for model of NT-4 function in early gangliogenesis to direct precursor cells along a BDNF-dependent lineage. In the absence of NT-4, precursors continue to expand and ultimately differentiate along the NT-3-dependent proprioceptive pathway.

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