We report that the neurotrophin receptor TrkB is expressed in a subset of embryonic sympathoblasts during the early development of lumbar paravertebral sympathetic ganglia in chicken and mouse embryos. In the chicken, TrkB expression is transient, and completely lost by St 34 (E8). Since BDNF induces the proliferation of sympathoblasts in cell culture, yet in vivo there is little proliferation observed in TrkB-positive cells in nascent ganglia, we propose that if TrkB activation becomes unregulated by excess BDNF or constitutive phosphorylation of TrkB , this transient population of TrkB-positive sympathoblasts may trigger the genesis of neuroblastoma, a childhood tumor found in the paravertebral chain and adrenal medulla.
The two populations of sympathoblasts that we observe support previous findings of heterogeneity among developing sympathetic neurons and neural crest cells. Early sympathetic ganglia contain at least two subpopulations: early differentiating neurons that lack TrkB expression and express TrkA and TrkC, and late differentiating sympathoblasts that express TrkB. Explant cultures of sympathetic ganglia from E16 chick embryos give rise to two neuronal populations: one that remains close to the explant, and one that migrates away from the explant . In addition, early neuronal subpopulations have been observed in cultures of neural crest cells from St. 13/14 quail embryos as evidenced by the expression of neuronal cell type-specific gangliosides . Perhaps these different subpopulations will ultimately give rise to the two neurochemically distinct populations found in lumbar sympathetic ganglia: the noradrenergic, NPY-containing neurons that innervate internal organs and enteric ganglia and the cholinergic, VIP-containing neurons that innervate vasculature in the hind limbs.
The effects of BDNF and TrkB deletion and over expression have been studied on superior cervical ganglion and preganglionic neurons in thoracic segments of the spinal column, but not on paravertebral sympathetic neurons. In the superior cervical ganglion, an increase in the number of neurons of BDNF null mice is likely due to apoptosis induced by BDNF via p75NTR . In contrast, the responses of paravertebral sympathetic neurons to BDNF are complex and subtype dependent. Over expression of BDNF leads to an increase in the number of noradrenergic fibers innervating the erector pilli muscles of hair follicles, while noradrenergic fibers innervating blood vessels were unaffected . If our results indicating that BDNF promotes proliferation of TrkB-positive sympathoblasts in the chicken embryo can be extrapolated to the subset of TrkB-positive sympathoblasts in murine ganglia, then these TrkB-positive cells may be neurons destined to innervate the erector pilli. In other studies, TrkB null mice showed no changes in morphology or cell number in superior cervical ganglia  or in the intermediolateral column ; but this may not be predictive of a phenotype in the lumbar paravertebral chain. It is thus possible that BDNF/TrkB signaling could play a specific role in other regions of the paravertebral sympathetic chain, such as the lumbar region. However, if TrkB-positive cells are not normally actively proliferating in vivo, then it would not be surprising that the development of the paravertebral sympathetic chain is not disrupted in TrkB or BDNF null mice. It may be more informative to examine mice that over express BDNF on a promoter that targets expression to embryonic lumbar ganglia. Unfortunately, such mice do not exist.
Our findings that the St. 29/30 (E6.5) sympathoblasts are dependent on both NT-3 and NGF for survival in culture are consistent with previous work on mouse sympathoblasts from the superior cervical ganglion . In these studies, NT-3 and NGF deletion separately led to a decrease in the number of sympathetic neurons at E17.5 compared to control. Deletion of both NT-3 and NGF together did not enhance cell death. In contrast, cultured rat superior cervical ganglion sympathetic neurons respond to NT-3 at E14.5 and then to NGF at E19.5, although time points in between were not analyzed .
In addition to promoting survival, NT-3, NGF, and BDNF also induce proliferation of various neuronal precursors at different stages of development. NT-3 can promote the incorporation of [3H]-thymidine into cultured quail neural crest cells from the trunk region [21, 22], Later in rat sympathetic development, NT-3 supports survival of neurons, but does not promote proliferation , which is consistent with our results. NGF promotes an increase in BrdU incorporation from 25% to 35% in the DRG cervical segment 2 in the chick embryo . In chicken embryos that are treated with NGF in ovo at St. 18 and 21, there is an increase in BrdU uptake after formation of the primary sympathetic chain at St. 23 . Since NGF does not appear to affect proliferation of St. 29/30 (E6.5) chick sympathoblasts, NGF may only promote proliferation in primary, but not secondary chain sympathoblasts. Motor neuron progenitors in the ventral neural tube from the chick embryo express TrkB and when ventral neural tube explants are treated with BDNF, there is an increase in the number of motor neurons produced and BrdU incorporation . BDNF also promotes the proliferation of cultured neuroblastoma cells . Taken together, these results are consistent with our findings that NT-3 and NGF do not promote proliferation of St. 29/30 (E6.5) sympathoblasts, and support the assertion that BDNF promotes proliferation of TrkB-positive sympathoblasts in culture.
Our observations suggest a transient function of TrkB during early sympathetic development in supporting proliferation of this early subpopulation of sympathoblasts. However, the in vivo labeling suggests that only a minority (10–20%) of this population is dividing during the window that TrkB is expressed. In light of the very strong proliferative effect produced in cell culture, these TrkB expressing cells could respond more strongly if endogenous BDNF rises to higher levels, or if the mechanism that down regulates TrkB expression becomes nonfunctional. Such events could trigger an early proliferative event that leads to a cascade of changes that initiates transformation of cells to neuroblastoma. Thus, these early TrkB expressing cells help solve the puzzle as to why TrkB is expressed in aggressive and invasive forms of neuroblastoma, particularly because BDNF induces cultured neuroblastoma cells to become more proliferative, invasive, angiogenic, and resistant to chemotherapeutic reagents than untreated cultures . Future studies will determine whether constitutive expression of BDNF and TrkB in the chick embryo sustains proliferation of differentiating sympathoblasts.