Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors

Abstract

To understand mechanisms resulting in the absence of two-thirds of spinal sensory neurons in mice lacking NT-3, we have compared dorsal root ganglia development in normal and mutant embryos. The reduction in neurons, achieved by E13, results from several deficits: first, elevated neuronal apoptosis significantly reduces neuronal numbers; second, elevated neurogenesis between E11 and E12, without changes in rates of precursor proliferation or apoptosis, depletes the precursor pool; consequently, the reduced precursor pool prevents increases in neuronal numbers between E12 and E13, when most neurons are born in normal animals. Although deficits occur before final target innervation, we show that NT-3 is expressed at all stages in regions accessible to these neurons or their axons and is only restricted to final targets after innervation.

Figure 1

Analysis and Phenotype of Early DRG Development in Wild-Type and NT-3 Mutant Mice (A and B) Transverse sections through a wild-type ([A], +/+) and a mutant ([B], −/−) embryo at embryonic day (E) 9 stained with antibodies to the low affinity neurotrophin receptor p75^NTR to label neural crest cells (arrows) migrating out of the neural tube (nt). Neural crest migration is apparently normal in embryos deficient for NT-3 (cells are also labeled within the ventral neural tube). (C and D) Thoracic DRGs as seen in sagittal sections through E10 embryos stained either with p75^NTR antibodies and counterstained with cresyl violet (C) or with antibodies to the 150 KDa neurofilament subunit (D). Neurofilament antibodies label the cell bodies and the projections of neurons present in the ganglia. dr, dorsal root. At this stage there is no difference between wild-type and mutant ganglia, and only mutants are shown. Note that neurons (neurofilament-positive cells) can not be distinguished from other cells in Nissl stained material. (E and F) L1 DRGs from wild-type (+/+) and mutant (−/−) embryos at E11 stained with cresyl violet. Note that the mutant ganglion is not significantly reduced in size but shows more pyknotic figures. (G and H) Nissl stained L1 DRGs from wild-type (+/+) and mutant (−/−) embryos at E13. The mutant ganglion shows a clear reduction in volume and more pyknotic figures. (I and J) L1 DRGs from wild-type (+/+) and mutant (−/−) embryos at E13 stained with anti-neurofilament antibodies, showing the same reduction in size for the mutant as in (G) and (H). Bars, 100 µm (A and B); 100 µm (C–J).

Figure 2

Graphical Representations of Numbers of Cells, Neurons, and Precursors in Normal and Mutant DRGs (A) Quantitation of the number of total cells and neurons at different embryonic (E) and postnatal (P) stages in thoracic 1 (T1) and lumbar 1 (L1) DRGs in wild types and NT-3 mutants. Total number of cells (circles) and of neurons (squares) in wild-type (filled symbols, continuous line) and mutant (empty symbols, dotted line) ganglia. Numerical data used to construct these graphs is presented in Tables 1 and 2. Since the normal development and phenotype of the mutation are similar in all ganglia analyzed, the data for only two of the four ganglia are presented graphically here. At E10 there are no differences between wild-type and mutant embryos in the number of cells or neurons, as seen in the T1 DRG (lumbar ganglia are not well defined at this stage). At E13, the final number of neurons in normal embryos and the complete deficit found in mutant embryos at birth are achieved in both T1 and L1 DRGs. Note that the number of neurons is significantly reduced in mutant ganglia at E11 (see Table 1 for statistical significance), but that between E11 and E12 higher than normal neurogenesis in the mutant eliminates the deficits in total neuronal numbers by E12. Between E12 and E13, the number of neurons increases very rapidly in wild-type but not in mutant animals. (B) Relative percentages of cells, neurons, and precursors (total number of cells minus number of neurofilament positive cells) present in mutant compared with wild-type ganglia from E10 to E13. Data from the four different ganglia analyzed are plotted: T1 (squares), T6 (diamonds), L1 (circles), and L4 (triangles). There is an initial significant (see Table 1) deficit in the number of neurons in all ganglia at E11 that is compensated at E12. In lumbar ganglia, a deficit in neurons is not accompanied by a deficit in precursors. Notice also that the increased numbers of neurons appearing in mutant ganglia between E11 and E12 is accompanied by equally dramatic reductions in the numbers of precursors, suggesting that neurogenesis may be accelerated in the absence of NT-3.

Figure 3

Proliferation and Cell Death in Wild-Type (+/+) and Mutant (−/−) DRG (A–F) Illustrations of assays of proliferation and apoptosis in T1 DRGs of E11 embryos. (A and B) Immunodetection of BrdU after a 2 hr pulse in E11 wild-type (A) and mutant (B) embryos. Notice that BrdU-positive nuclei are not pyknotic. (C and D) High magnification micrographs of Nissl stained sections through ganglia of E11 wild-type (C) and mutant (D) embryos. At this stage, pyknotic figures (arrows) and mitotic figures (empty arrows) can be observed. Notice that pyknotic profiles are more frequent in the mutant ganglion. Care was taken to exclude red blood cells (arrowhead) from the quantitation. (E and F) Sections of ganglia of E11 wild-type (E) and mutant (F) embryos stained with the TUNEL method for the detection of cells undergoing apoptosis. In agreement with an increase in pyknotic figures, the mutant ganglion shows more cells stained by this method. Bars, 100 µm (A and B); 50 µm (C and D); 100 µm (E and F). (G–I) Quantification of apoptosis and proliferation in wild-type and mutant DRGs. (G) Quantitation of the rate of cell death in wild-type (+/+, solid bars) and mutant (−/−, shaded bars) ganglia at stage E11 for thoracic 1 (T1), thoracic 6 (T6), lumbar 1 (L1), and lumbar 4 (L4) DRGs. Dying cells were calculated by counting all pyknotic figures in each of the different ganglia. The numbers are expressed as the relative number of dying cells per total number of cells present in each ganglion. The number of embryos analyzed in each case are the same as in Table 2. (H) Cell death rates between E11 and E17 in wild-type (filled symbols, continuous line) and mutant (unfilled symbols, dotted line) embryos, obtained by dividing the number of pyknotic figures either by the total number of cells (E11-E13) or by the total number of neurons (E15-E17) present in the ganglia (see Tables 1 and 2). At E10 there are no pyknotic figures. Represented are the values for T1 (squares) and L1 (circles) DRGs as examples. Very similar data was obtained for T6 and L4 DRGs, but are not presented for simplicity. Notice that cell death is significantly elevated in the mutant ganglia at all stages between E11 and E13. At E15 and E17, cell death in the ganglia of both wild-type and mutant animals is very low and is not elevated significantly in mutant ganglia. (I) Quantitation of the rate of BrdU incorporation in wild-type (+/+, solid bars) and mutant (−/−, shaded bars) ganglia at E11. The rate of BrdU incorporation or labeling index was calculated by dividing the number of BrdU-positive nuclei in each ganglion by the calculated number of precursors (see also Table 3). No significant differences in labeling index were found comparing wild-type and mutant ganglia. In each case, values are represented as mean ± SEM. Statistical significance was tested using a one-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Proliferation and Cell Death in Wild-Type (+/+) and Mutant (−/−) DRG (A–F) Illustrations of assays of proliferation and apoptosis in T1 DRGs of E11 embryos. (A and B) Immunodetection of BrdU after a 2 hr pulse in E11 wild-type (A) and mutant (B) embryos. Notice that BrdU-positive nuclei are not pyknotic. (C and D) High magnification micrographs of Nissl stained sections through ganglia of E11 wild-type (C) and mutant (D) embryos. At this stage, pyknotic figures (arrows) and mitotic figures (empty arrows) can be observed. Notice that pyknotic profiles are more frequent in the mutant ganglion. Care was taken to exclude red blood cells (arrowhead) from the quantitation. (E and F) Sections of ganglia of E11 wild-type (E) and mutant (F) embryos stained with the TUNEL method for the detection of cells undergoing apoptosis. In agreement with an increase in pyknotic figures, the mutant ganglion shows more cells stained by this method. Bars, 100 µm (A and B); 50 µm (C and D); 100 µm (E and F). (G–I) Quantification of apoptosis and proliferation in wild-type and mutant DRGs. (G) Quantitation of the rate of cell death in wild-type (+/+, solid bars) and mutant (−/−, shaded bars) ganglia at stage E11 for thoracic 1 (T1), thoracic 6 (T6), lumbar 1 (L1), and lumbar 4 (L4) DRGs. Dying cells were calculated by counting all pyknotic figures in each of the different ganglia. The numbers are expressed as the relative number of dying cells per total number of cells present in each ganglion. The number of embryos analyzed in each case are the same as in Table 2. (H) Cell death rates between E11 and E17 in wild-type (filled symbols, continuous line) and mutant (unfilled symbols, dotted line) embryos, obtained by dividing the number of pyknotic figures either by the total number of cells (E11-E13) or by the total number of neurons (E15-E17) present in the ganglia (see Tables 1 and 2). At E10 there are no pyknotic figures. Represented are the values for T1 (squares) and L1 (circles) DRGs as examples. Very similar data was obtained for T6 and L4 DRGs, but are not presented for simplicity. Notice that cell death is significantly elevated in the mutant ganglia at all stages between E11 and E13. At E15 and E17, cell death in the ganglia of both wild-type and mutant animals is very low and is not elevated significantly in mutant ganglia. (I) Quantitation of the rate of BrdU incorporation in wild-type (+/+, solid bars) and mutant (−/−, shaded bars) ganglia at E11. The rate of BrdU incorporation or labeling index was calculated by dividing the number of BrdU-positive nuclei in each ganglion by the calculated number of precursors (see also Table 3). No significant differences in labeling index were found comparing wild-type and mutant ganglia. In each case, values are represented as mean ± SEM. Statistical significance was tested using a one-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Attempts to Detect Apoptosis in Neurons and Neuronal Precursors (A) Double immunofluorescence labeling for TUNEL (green) and neurofilament (NF150, red) in a thoracic ganglion of an E11 mutant embryo. In this figure, there are two immunopositive cells for neurofilament in the field whose nuclei are labeled by the apoptosis detection method (arrows). This suggests that neurons die at this stage. (B and C) Double labeling for TUNEL (green) and BrdU (red) after either 2 or 5 hr injection pulse of BrdU at E11 in thoracic ganglia, respectively. Colocalization was never observed, indicating that precursors are not dying in the mutant animals, but instead are lost through more rapid differentiation into cells with a neuronal phenotype. Bars, 10 µm (A and C); 10 µm (B).

Figure 5

Development of NT-3 Expression and Axonal Projections in E10-E15 Embryos as Assessed by the Expression of the Reporter Gene lacZ Introduced into the NT-3 Locus and β-III-Tubulin Heterozygous mice were histochemically stained for β-galactosidase activity either in whole mounts or in sections through the thoracic region. (A) E10 embryo double-stained for β-galactosidase (blue) and β-III-tubulin (brown) to show the relationship between the expression of the reporter and the developing nervous system. LacZ expression is observed in the head, including the top of the mesencephalon (m), the branchial arches, the developing ear and eye, and in the anterior part of the trunk. Staining is also observed in the most proximal part of the forelimb and, more caudally, in the dorsal aorta (da). Antibodies to tubulin label all neurons and their projections, including all cranial ganglia (V, VIII, IX–X) and all formed DRGs (drg). Notice that DRGs are more developed rostrally, the caudal DRGs being barely discernible at this stage. c, cervical; t, thoracic; l, lumbar. Note also that the expression of the lacZ reporter is not within the DRGs themselves but in the surrounding area, especially around the projecting axons. (B) At E11 the expression becomes more intense and extends caudally. Conspicuous staining is now seen in the proximal part of both forelimb (fl) and hindlimb (hl). (C) Dorsal view of an E11 embryo showing two longitudinal stripes of intense staining that correspond to the lateral motor columns (lmc). The staining is particularly strong at the level of the forelimb (fl) (empty arrows). (D) Higher magnification of a forelimb (fl) of an E11 embryo double-labeled for β-galactosidase and β-III-tubulin, showing that the growing peripheral projection is exposed to high levels of NT-3. hl, hindlimb. (E) Transverse vibratome section through the thoracic region of the E10 embryo shown in (A). Expression of the lacZ reporter is seen lateral to the DRGs (drg) and strongly around the tip of the projecting nerve (n). Additional staining is seen in the developing lateral motor column (lmc) and in the forelimb (fl). (F) Transverse vibrotome section of an E11 embryo containing one side of the spinal cord and body (bo) and one forelimb (fl). The histochemical reaction was combined with immunocytochemistry with antibodies to neurofilament to label the DRGs (drg) and growing axons. Intense staining for β-galactosidase is seen in areas where sensory and motor axons are actively growing. Especially strong is the expression surrounding the branch projecting dorsally toward the skin (arrowhead), close to the DRG. The most distal part of the limb, devoid of axons at this time, does not express NT-3 (delineated by open arrows). The staining in the lateral motor column (lmc) is particularly strong at this stage. bo, body. (G) Double-stained cryosection through the forelimb of an embryo at E13 showing that both axons (n) and lacZ expression have extended more distally (as far as broken arrows). Simultaneously, expression of the reporter is downregulated in the body (bo) becoming restricted to some muscles (mu) and to the skin (s). c, cartilage. (H) Double-stained cryosection through the most distal part of a forelimb of an E15 embryo showing that simultaneous labeling for axonal projections and reporter expression occurs at the tip of the limb. More proximally, expression is strongly down-regulated. bo, body; c, cartilage; di, digit. Bars, 1 mm (A–D); 0.5 mm (E and H).

Figure 6

Expression of lacZ and Neurofilament in Sections of Developing Heterozygous Embryos at the Stages Indicated to Show the Relationship between Growing Peripheral Axons and NT-3 Expression (A) Transverse section through the thoracic region of an E11 embryo showing the spinal cord, DRGs (drg), and the peripheral nerves (n). Strong expression of the reporter is found in the ventral horn of the spinal cord, where the lateral motor column (lmc) is developing, and around the DRG and sensory-motor projection. The skin (s) expresses low, if detectable NT-3 at this stage. Notice that there is no expression of NT-3 within the DRGs themselves. (B) Peripheral axons at the mid part of the forelimb. Axons growing through areas of mesenchymal/pre-muscle cells (ms) are surrounded by cells expressing NT-3, as assessed by lacZ expression, while the expression decreases dramatically toward the distal end of the limb (to the right), where axons have not reached. The epidermis (e), also devoid of any innervation at this time, does not express NT-3. (C) Expression of NT-3, as assessed by lacZ, by neurons within the DRGs is not seen until E15 when a small number of cells express this factor. (D) At E13, when sensory axons invade profusely the epidermis (e), NT-3 expression is strong there, but disappears from the mesenchyme (ms). (E) Expression of the reporter in formed skeletal muscle as seen at E17. The neurotrophin appears to be produced in fetal myoblasts (arrows), but not in muscle fibers. (F and G) Late expression in the skin becomes restricted to hair follicles (hf), as can be seen either in whole mounts of skin (F) or in sections (G). Expression is also seen in the basal layer of the epidermis (b). p, papilla; mu, muscle. Bars, 20 µm (A); 100 µm (C); 100 µm (B, D, E, and F).