Heterogeneous requirement of NGF for sympathetic target innervation in vivo

Abstract

The neurotrophin nerve growth factor (NGF) plays a crucial role in the development of the sympathetic nervous system. In addition to being required for sympathetic neuron survival in vivo and in vitro, NGF has been shown to mediate axon growth in vitro. The role of NGF in sympathetic axon growth in vivo, however, is not clear because of its requirement for survival. This requirement can be circumvented by a concomitant deletion of Bax, a pro-apoptotic Bcl-2 family member, thus allowing an examination of the role of neurotrophins in axon growth independently of their function in cell survival. Here, we analyzed peripheral sympathetic target organ innervation in mice deficient for both NGF and Bax. In neonatal NGF-/-; Bax-/- mice, sympathetic target innervation was absent in certain organs (such as salivary glands), greatly reduced in others (such as heart), somewhat diminished in a few (such as stomach and kidneys), but not significantly different from control in some (such as trachea). At embryonic day 16.5, peripheral target sympathetic innervation was also reduced in NGF-/-; Bax-/- mice, with analogous variability for different organs. Interestingly, in some organs such as the spleen the precise location at which sympathetic axons become NGF-dependent for growth was evident. We thus show that NGF is required for complete peripheral innervation of both paravertebral and prevertebral sympathetic ganglia targets in vivo independently of its requirement for cell survival. Remarkably, target organs vary widely in their individual NGF requirements for sympathetic innervation.

Figure 1.

Elimination ofBax preserves sympathetic chain ganglia neurons in the absence of NGF. A-H, Whole-mount tyrosine hydroxylase immunostaining of E16.5 (A-D) and P0.5 (E-H) sympathetic chain from stellate ganglion (arrows) to T3 chain ganglion (A-D) and from T1 ganglion (arrowheads) to T3 ganglion (E-H). NGF^+/+; Bax^-/-mice (C, G) serve as controls for NGF^-/-; Bax^-/-mice (D, H), showing no significant effects of removal of NGF in the Bax^-/- background. This is in contrast to NGF^-/-; Bax^+/+ mice (F), which demonstrate a clear gross reduction in the size of sympathetic chain ganglia compared with NGF^-/-; Bax^-/- mice (H), indicating the effects of deleting Bax in the NGF^-/- background on sympathetic chain ganglia cell preservation. NGF^+/+; Bax^+/+ mice (A, E) are also included as controls. Note that proximal sympathetic chain ganglia projections are preserved at both ages and in all genotypes (A-H). The number of animals examined in this and subsequent experiments is as follows: at E16.5, n = 3 (NGF^+/+; Bax^+/+), 5 (NGF^-/-; Bax^+/+), 4 (NGF^+/+; Bax^-/-), 3 (NGF^-/-; Bax^-/-); at P0.5, n = 6 (NGF^+/+; Bax^+/+), 5 (NGF^-/-; Bax^+/+), 3 (NGF^+/+; Bax^-/-),4(NGF^-/-;Bax^-/-). Scale bar: (shown in A) A-H, 100 μm. I, Number of SCG neurons at P0.5. Shown are means ± SEM of neuronal counts in three animals for each genotype. ^*p < 0.0001 (Significantly different from all other groups); ^** indicates not statistically different from one another, as determined using one-way ANOVA followed by Bonferroni/Dunn post hoc test.

Figure 2.

SCG neurons extend proximal axonal projections but fail to innervate their target organs in NGF^-/-; Bax^-/- mice. A, B, Whole-mount tyrosine hydroxylase immunostaining of E16.5 SCG (arrows) and its proximal projections demonstrates that SCG neurons extend proximal projections in NGF^-/-; Bax^-/-mice (B) as compared with NGF^+/+; Bax^-/- controls (A). C-F, Whole-mount tyrosine hydroxylase immunostaining of submaxillary salivary gland indicates that sympathetic neuron projections along blood vessels entering the gland (arrowheads) and innervation of the parenchyma are absent in NGF^-/-; Bax^-/- mice (D, F) as compared with NGF^+/+; Bax^-/- controls (C, E) at both E16.5 (C, D) and P0.5 (E, F). G, H, Whole-mount tyrosine hydroxylase immunostaining of cutaneous sympathetic innervation of the eye at E16.5 indicates that SCG neurons fail to innervate this target in NGF^-/-; Bax^-/- mice (H) as compared with control NGF^+/+; Bax^-/- animals (G). Scale bars: A-D, 100 μm; E-H, 250 μm.

Figure 3.

Sympathetic innervation of the heart is deficient but that of the trachea is maintained in NGF^-/-; Bax^-/- mice. A-D, Whole-mount tyrosine hydroxylase immunostaining of heart ventricles (arrows) shows the lack of sympathetic innervation in NGF^-/-; Bax^-/- mice (B, D) as compared with NGF^+/+; Bax^-/- controls (A, C) at both E16.5 (A, B) and P0.5 (C, D). E-H, Whole-mount tyrosine hydroxylase immunostaining of dorsal trachea reveals a slight reduction in its sympathetic innervation at E16.5 (E, F) but no significant gross differences in the level of its sympathetic innervation at P0.5 (G, H)in NGF^-/-; Bax^-/- mice (F, H) as compared with NGF^+/+; Bax^-/- controls (E, G). Scale bars: A, B, E, F, 100 μm; C, D, G, H, 250 μm.

Figure 4.

NGF is required for complete sympathetic innervation of the stomach. A-H, Whole-mount tyrosine hydroxylase immunostaining of body of the stomach demonstrates deficient sympathetic innervation in NGF^-/-; Bax^-/- mice (D, H) as compared with NGF^+/+; Bax^-/- controls (C, G) at both E16.5 (C, D) and P0.5 (G, H). Lower magnification photographs illustrate presence of sympathetic innervation in the cardiac region of the stomach (arrows) for all genotypes at E16.5 (A, B) and P0.5 (E, F). Although shown at the same magnification, NGF^+/+; Bax^-/- stomach at P0.5 (E) appears larger than NGF^-/-; Bax^-/- stomach at P0.5 (F) because of distension from milk in control stomach, which is absent in NGF^-/-; Bax^-/- animals because of deficient feeding behavior in the absence of NGF. Scale bars: A-D, 100 μm; E-H, 250 μm.

Figure 5.

The kidney is incompletely innervated by sympathetic neurons in NGF^-/-; Bax^-/- mice. A-F, Whole-mount tyrosine hydroxylase immunostaining of kidneys reveals the presence of sympathetic axon extension along renal arteries (A-D, arrows) but deficient innervation of kidney parenchyma (E, F) in NGF^-/-; Bax^-/- mice (B, D, F) as compared with NGF^+/+; Bax^-/-controls (A, C, E) at both E16.5 (A, B) and P0.5 (C-F). Scale bars: A, B, E, F, 100 μm; C, D, 250 μm.

Figure 6.

Sympathetic innervation of the gastrointestinal tract is deficient in NGF^-/-; Bax^-/- mice. A-J, Whole-mount tyrosine hydroxylase immunostaining of the entire E16.5 gastrointestinal tract shows that mesenteric sympathetic axon projections (arrows) are present but diminished in NGF^-/-; Bax^-/- mice (B) as compared with NGF^+/+; Bax^-/- controls (A). Closer inspection of the proximal small intestine (C-F) demonstrates that sympathetic axons extend along the vasculature toward the small intestine (arrowheads) but do not fully innervate the organ in NGF^-/-; Bax^-/- mice (D, F) as compared with NGF^+/+; Bax^-/- controls (C, E) at both E16.5 (C, D) and P0.5 (E, F). The distal large intestine (G-J) also shows a reduction in sympathetic fiber density in NGF^-/-; Bax^-/- mice (H, J) as compared with NGF^+/+; Bax^-/- controls (G, I) at both E16.5 (G, H) and P0.5 (I, J). Scale bars: A, B, E, F, I, J, 250 μm; C, D, G, H, 100 μm.

Figure 7.

NGF is required for complete sympathetic innervation of ureters, bladder, and gonads. A-D, Whole-mount tyrosine hydroxylase immunostaining of proximal ureters reveals a deficit in sympathetic innervation in NGF^-/-; Bax^-/- mice (B, D) as compared with NGF^+/+; Bax^-/- controls (A, C) at both E16.5 (A, B) and P0.5 (C, D). E, F, Whole-mount tyrosine hydroxylase immunostaining of E16.5 bladder shows a reduction in sympathetic innervation in NGF^-/-; Bax^-/- mice (F) as compared with NGF^+/+; Bax^-/- controls (E). G-J, Whole-mount tyrosine hydroxylase immunostaining of gonads demonstrates a deficiency in sympathetic innervation in NGF^-/-; Bax^-/- mice (H, J) as compared with NGF^+/+; Bax^-/- controls (G, I) at both E16.5 (G, H) and P0.5 (I, J). Arrow indicates testis. Scale bars: A-H, 100 μm; I, J, 250 μm.

Figure 8.

Sympathetic axons extend along the lienal artery but fail to enter spleen parenchyma in NGF^-/-; Bax^-/- mice. A-D, Whole-mount tyrosine hydroxylase immunostaining of spleen reveals that sympathetic axons arrest at the entrance of the lienal artery into spleen parenchyma (arrow) in NGF^-/-; Bax^-/- mice (B, D) as compared with NGF^+/+; Bax^-/- controls (A, C) at both E16.5 (A, B) and P0.5 (C, D). Scale bars: A, B, 50 μm; C, D, 100 μm.