Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia

The molecular mechanisms responsible for specifying the dorsal-ventral pattern of neuronal identities in dorsal root ganglia (DRG) are unclear. Here we demonstrate that Sonic hedgehog (Shh) contributes to patterning early DRG cells. In vitro, Shh increases both proliferation and programmed cell death (PCD). Increasing Shh in vivo enhances PCD in dorsal DRG, while inducing greater proliferation ventrally. In such animals, markers characteristic of ventral sensory neurons are expanded to more dorsal positions. Conversely, reducing Shh function results in decreased proliferation of progenitors in the ventral region and decreased expression of the ventral marker trkC. Later arising trkA⁺ afferents make significant pathfinding errors in animals with reduced Shh function, suggesting that accurate navigation of later arising growth cones requires either Shh itself or early arising, Shh-dependent afferents. These results indicate that Shh can regulate both cell number and the distribution of cell types in DRG, thereby playing an important role in the specification, patterning and pathfinding of sensory neurons.     

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target‐derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret‐expressing neurons in the DRG complete developmental cell death prior to TrkA‐expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701–717, 2018     

Axial level-dependent differences in size of avian dorsal root ganglia are present from gangliogenesis

Dorsal root ganglia (DRG) form from neural crest cells that colonize the rostral sclerotomes of the somites of the trunk of higher vertebrates. Differences in sensory field size and innervation density are reflected by the size of the ganglia at different axial levels. Some of the position-dependent differences in ganglionic size derives from asymmetry in embryonic programmed cell death, when more DRG neurons die at non-limb-innervating levels than at the levels that innervate the limbs. I have now examined early chick embryos determine whether there is asymmetry in DRG size at the time of their condensation at stage 20 [embryonic day (E) 3], before the onset of cell death. Ganglia in brachial segments 14 and 15 are more than 80% larger on average than those in cervical segments 5 and 6 at this stage of development. This difference in volume is due to increased numbers of cells in the brachial sensory ganglia. Several other morphometric parameters of the DRG and sclerotomes were then determined. The rostro-caudal length was found to be significantly greater for brachial ganglia. The greater length of the brachial ganglia was found to be correlated with (1) a greater length of brachial than cervical sclerotomes and (2) the occupation by brachial ganglia of a larger proportion of the rostro-caudal extent of the sclerotome. These results demonstrate that the mature pattern of axial differences in ganglionic size are foreshadowed by a pattern set during the period of gangliogenesis, which is then further sculpted by apoptosis. This initial axial asymmetry in size preceding cell death parallels that recently shown for the development of the ventral motor column. Thus the sensory ganglia, like the somites, have distinct axial-position-dependent characteristics from their outset, characteristics that are not dependent on cell death resulting from competition for target-derived survival factors as was previously thought. DRG positional identities, like their segmentation, may be imposed by the paraxial mesoderm or may alternatively be intrinsically determined as is the case for the rhombomeres of the hindbrain. © 1993 John Wiley & Sons, Inc.     

Zebrafish Rohon-Beard Neuron Development: Cdk5 in the Midst

Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase that is activated mostly by association with its activators, p35 and p39. Initially projected as a neuron-specific kinase, cdk5 is expressed ubiquitously and its kinase activity solely depends on the presence of its activators, which are also found in some non-neuronal tissues. As a multifunctional protein, cdk5 has been linked to axonogenesis, cell migration, exocytosis, neuronal differentiation and apoptosis. Cdk5 plays a critical role in functions other than normal physiology, especially in neurodegeneration. Its contribution to both normal physiological as well as pathological processes is mediated by its specific substrates. Cdk5-null mice are embryonically lethal, therefore making it difficult to study precisely what cdk5 does to the nervous system at early stages of development, be it neuron development or programmed cell death. Zebrafish model system bypasses the impediment, as it is amenable to reverse genetics studies. One of the functions that we have followed for the cdk5 ortholog in zebrafish in vivo is its effect on the Rohon-Beard (RB) neurons. RB neurons are the primary sensory spinal neurons that die during the first two days of zebrafish development eventually to be replaced by the dorsal root ganglia (DRG). Based on ours studies and others’, here we discuss possible mechanisms that may be involved in cdk5’s role in RB neuron development and survival.     

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 ras

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors, TrkB and TrkC. In contrast to BDNF, NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLCγ were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons. Special issue dedicated to Dr. Hans Thoenen.     

NT-3 and CNTF exert dose-dependent, pleiotropic effects on cells in the immature dorsal root ganglion: neuregulin-mediated proliferation of progenitor cells and neuronal differentiation

Neurons in the nascent dorsal root ganglia are born and differentiate in a complex cellular milieu composed of postmitotic neurons, and mitotically active glial and neural progenitor cells. Neurotrophic factors such as NT-3 are critically important for promoting the survival of postmitotic neurons in the DRG. However, the factors that regulate earlier events in the development of the DRG such as the mitogenesis of DRG progenitor cells and the differentiation of neurons are less defined. Here we demonstrate that both NT-3 and CNTF induce distinct dose-dependent responses on cells in the immature DRG: at low concentrations, they induce the proliferation of progenitor cells while at higher concentrations they promote neuronal differentiation. Furthermore, the mitogenic response is indirect; that is, NT-3 and CNTF first bind to nascent neurons in the DRG--which then stimulates those neurons to release mitogenic factors including neuregulin. Blockade of this endogenous neuregulin activity completely blocks the CNTF-induced proliferation and reduces about half of the NT-3-mediated proliferation. Thus, the genesis and differentiation of neurons and glia in the DRG are dependent upon reciprocal interactions among nascent neurons, glia, and mitotically active progenitor cells.     

Neurotrophins and cell death

The neurotrophins - NGF, BDNF, NT-3 - are secreted proteins that play a major role in neuron survival, differentiation and axon wiring toward target territories. They do so by interacting with their main tyrosine kinase receptors TrkA, TrkB, TrkC and p75(NTR). Even though there is a general consensus on the view that neurotrophins are survival factors, there are two fundamentally different views on how they achieve this survival activity. One prevailing view is that all neurons and more generally all normal cells are naturally committed to die unless a survival factor blocks this death. This death results from the engagement of a "default" apoptotic cell program. The minority report supports, on the opposite, that neurotrophin withdrawal is associated with an active signal of cell death induced by unbound dependence receptors. We will discuss here how neurotrophins regulate cell death and survival and how this has implications not only during nervous system development but also during cancer progression.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT- 3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross- sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.     

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.     

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.     

Expression of neurotrophin receptors trkB and trkC and their ligands in rat adrenal gland and the intermediolateral column of the spinal cord

Neurotrophins and their trk receptors constitute major classes of signaling molecules with important actions in the developing and adult nervous system. With regard to the sympathoadrenal cell lineage, which gives rise to sympathetic neurons and chromaffin cells, neurotrophin-3 (NT-3) and nerve growth factor (NGF) are thought to influence developing sympathetic neurons. Neurotrophin requirements of chromaffin cells of the adrenal medulla are less well understood than those for NGF. In order to provide the bases for understanding of putative functions of neurotrophins for the development and maintenance of chromaffin cells and their preganglionic innervation, in situ hybridization has been used to study the expression of brain-derived neurotrophic factor (BDNF) and NT-3, together with their cognate receptors trkB and trkC, in the adrenal gland and in the intermediolateral column (IML) of the spinal cord. BDNF is highly expressed in the embryonic adrenal cortex and later in cells of the cortical reticularis zone. Adrenal medullary chromaffin cells fail to express detectable levels of mRNAs for BDNF, NT-3, and their cognate receptors trkB and trkC. Neurons in the IML express BDNF and trkB, and low levels of NT-3 and trkC. Our data make it unlikely that BDNF and NT-3 serve as retrograde trophic factors for IML neurons but suggest roles of BDNF and NT-3 locally within the spinal cord and possibly for sensory nerves of the adrenal cortex.     

Dynamic expression of neurotrophin receptors during sensory neuron genesis and differentiation

To identify potential functions for neurotrophins during sensory neuron genesis and differentiation, we determined the temporal and spatial protein expression patterns of neurotrophin receptors throughout the process of sensory neurogenesis in the dorsal root ganglia (DRG). We show that neurotrophin receptors are expressed early, being first detected on subsets of migrating neural crest cells, and that trkC is among the earliest markers of neural lineage specification. In the immature DRG, we find that both trkC and p75(NTR) are expressed on subsets of dividing progenitor cells in vivo. Furthermore, our data directly reveal distinct patterns of trk receptor expression by individual sensory neurons from the time of their inception with all early arising cells initially being trkC(+), some subsets of whom also coexpress either trkA or trkB or both. As sensory neurons innervate their targets and establish their mature identities, the spectrum of trk receptors expressed by individual neurons is altered. The stereotyped trk receptor expression profiles identified here may potentially correspond to distinct lineages of sensory neurons. These data, in conjunction with other studies, argue for multiple functions for neurotrophins during the process of sensory neuron differentiation, including effects on both neural crest and DRG mitotically active progenitor cells, in addition to possibly influencing the establishment of sensory neuron identity.     

Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors.

Neurotrophins, such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), are potent regulators of neuronal functions. Here we show that human immune cells also produce NT-3 mRNA, secrete BDNF, and express their specific receptors trkB and trkC. The truncated trkB receptor, usually expressed in sensory neurons of the central nervous system, was also constitutively expressed in unstimulated Th cells. Full-length trkB was detectable in stimulated PBMC, B cell lines, and Th1, but not in Th2 and Th0 cell clones. Clonally restricted expression was also observed for trkC, until now not detected on blood cells. The Th1 cytokine IL-2 stimulated production of trkB mRNA but not of trkC, whereas the Th2 cytokine IL-4 enhanced NT-3 but not BDNF mRNA expression. Microbial Ags, which influence the Th1/Th2 balance, could therefore modulate the neurotrophic system and thereby affect neuronal synaptic activity of the central nervous system.