Neurotrophins support the development of diverse sensory axon morphologies

Abstract

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax-/- neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.

Fig. 1.

Sensory axon growth is rudimentary in the absence of neurotrophins. Photomicrographs of neuronal cultures plated at moderate densities (2000 neurons per well) and grown for 72 hr in the presence or absence of 50 ng/ml NGF are shown.a, Neurons from Bax+/+ mice extend long, fasciculated axons in the presence of NGF.b, Neurons from Bax−/− mice in the absence of neurotrophins (no NT) extend short, branched axons that do not form fascicles. c, In the presence of NGF, cultures from Bax−/− mice look indistinguishable from controls.

Fig. 2.

Quantification of neurotrophin responses.a–c, Photomicrographs show the typical pattern of axon extension from representative Bax−/− neurons grown for 72 hr in vitro without neurotrophins (a) or in the presence of 50 ng/mlNGF (b) or NT-3(c). d, e, Bar graphs show the percentage of neurons (± SEM) with one or two or more axons from Bax−/− mice and Bax+/+ littermates. Under these culture conditions, almost all neurons fromBax+/+ mice in the presence of either NGFor NT-3 were bipolar (stippled bar ind and wide-hatched bar ine, respectively). In contrast, in the absence of added neurotrophins (no NT), 90% ofBax−/− neurons were unipolar (open barsin d, e). Addition of eitherNGF (solid bars in d) orNT-3 (fine-hatched bars in e) resulted in a substantial fraction ofBax−/− neurons having two or more axons. Differences in percentages of neurons with a single axon between the no neurotrophin group and the groups treated with NGF orNT-3 were highly significant (p < 0.001; n = 6 embryos per group).

Fig. 3.

Morphological responses to NGF andNT-3. Camera lucida drawings of isolated DRG neurons from Bax−/− and Bax+/+ cultures after 72 hr in the absence of neurotrophins or in the presence of 50 ng/mlNGF or NT-3 are shown. Neurons with one (blue) or two or more (green andpink, respectively) axons were considered separately. Axons were arranged in order of increasing total length. A periodic sample of the entire population is shown for each condition.a, Bax−/− neurons cultured without neurotrophins (no NT) invariably had short highly branched axons. b, Addition of NGFresulted in the appearance of a subpopulation ofBax−/− neurons with much longer axons.c, Addition of NT-3 also resulted in extensive axon elongation, but note that the appearance ofNT-3–responsive neurons differed markedly from the appearance of those that responded to NGF.d, e, RepresentativeBax+/+ neurons from highly dissociated cultures in the presence of NGF (d) orNT-3 (e) are shown.

Fig. 4.

Quantification of the effects of neurotrophins on axon length. Total axon length was calculated by summing the lengths of all axons and branches. a,b, Each bar represents the mean ± SEM for six to seven E13 embryos. a,NGF. The open bar shows the total axon length of Bax−/− neurons in the absence of neurotrophins (no NT) after 72 hr. Unipolar neurons from Bax−/− mice treated withNGF (left solid bar) were not significantly longer than untreated neurons. Bax−/− neurons treated with NGF that were multipolar (right solid bar) showed a highly significant increase in total axon length (p < 0.001). These axons were not quite as long, however, as axons ofBax+/+ neurons in the presence of NGF(stippled bar). b, NT-3. Again, unipolar Bax−/− neurons in the presence ofNT-3 (left fine-hatched bar) were not significantly longer than Bax−/− neurons in the absence of neurotrophins (open bar). Multipolar neurons in the presence of NT-3 (right fine-hatched bar) showed a highly significant increase in total axon length (p < 0.001). For NT-3–responsive neurons, total length was comparable with the lengths of axons from Bax+/+ neurons (wide-hatched bar). c, Dose–response for axon growth at E13.Bax−/− neurons were grown in the absence (no NGF) or presence of increasing concentrations ofNGF for 1 d in vitro(DIV). Each point represents the mean total axon length (± SEM) measured from five embryos (20 neurons per embryo). Dosages of NGF as low as 0.5 ng/ml resulted in a significant increase in axon length compared with lengths from untreated neurons. Increasing concentrations of NGFenhanced the amount of axon outgrowth in a dose-dependent manner.d, Effects of neurotrophins on axon growth inE12 embryos. Each bar represents the mean ± SEM from five E12 embryos. The open bar shows the total axon length of Bax−/− neurons in the absence of neurotrophins (no NT) after 72 hr. Bax−/− neurons treated withNGF that demonstrated a morphological response (solid bar) showed a highly significant increase in total axon length (p < 0.001). Responsive neurons in the presence of NT-3(fine-hatched bar) also showed a highly significant increase in total axon length (p < 0.001).

Fig. 5.

NGF, NT-3, andBDNF mediate different morphological responses.a, d, g, j, Low-power photomicrographs (scale bar in j).b, c, e, f,h, i, k, l, High-power photomicrographs (scale bar in l). For visualizing morphologies of individual cells, neurons fromBax−/− mice were plated at low density (500 neurons per well) and grown for 72 hr in vitro without neurotrophins or in the presence of 50 ng/ml NGF,NT-3, or BDNF. a–c, Representative Bax−/− neurons cultured in the absence of neurotrophins (no NT). The neurons were unipolar and extended numerous short branches from the primary axon along its entire length. d–f, TypicalNGF-responsive Bax−/− neurons cultured in the presence of NGF. These neurons were bipolar and extended long, relatively unbranched axons. NGFtreatment suppressed the extension of short branches close to the cell soma. Typically, a few thick caliber branches were present along the distal portion of the axon. g–i, Typical NT-3-responsive Bax−/− neurons in the presence ofNT-3. NT-3 also appeared to suppress branching near the soma, although the primary axons of neurons treated with NT-3 were shorter than were those ofNGF-treated neurons. NT-3–responsive neurons almost invariably exhibited elaborate branching at the distal ends of their axons. j–l, RepresentativeBax−/− neurons depicting the characteristic morphological appearance of a subset of neurons that respond toBDNF. Note the prominent lathellipodia.

Fig. 6.

NGF and NT-3differentially regulate the extent of axonal branching.Bars show the mean number of branch points per E13 DRG neuron (± SEM) for six embryos. In the absence of neurotrophins (no NT), Bax−/− neurons had approximately five branch points per neuron after 72 hr in vitro. Treatment of Bax−/− andBax+/+ neurons with NGF resulted in a slight increase in the number of branch points per neuron. The presence of NT-3 resulted in a highly significant increase in the number of branch points per neuron (p < 0.001).