SHP-2 mediates target-regulated axonal termination and NGF-dependent neurite growth in sympathetic neurons

The tyrosine phosphatase SHP-2 has been implicated in a variety of signaling pathways, including those mediated by neurotrophins in neurons. To examine the role of SHP-2 in the development of sympathetic neurons, we inhibited the function of SHP-2 in transgenic mice by overexpressing a catalytically inactive SHP-2 mutant under the control of the human dopamine beta-hydroxylase promoter. Expression of mutant SHP-2 did not influence the survival, axon initiation, or pathfinding abilities of the sympathetic neurons. However, mutant SHP-2 expression resulted in an overproduction of sympathetic fibers in sympathetic target organs. This was due to interference with SHP-2 function, as overexpression of wild type SHP-2 had no such effect. In vitro, NGF-dependent neurite growth was inhibited in neurons expressing mutant SHP-2 but not in those expressing wild type SHP-2. Mutant (but not wt) SHP-2 expression also inhibited NGF-stimulated ERK activation. The NGF-dependent survival pathway was less affected than the neurite growth pathway. Our results suggest that NGF-regulated axon growth signals, and to a lesser degree survival signals, are mediated through a SHP-2-dependent pathway in sympathetic neurons. The increased sympathetic innervation in target tissues of neurons expressing mutant SHP-2 may result from interference with normal "stop" signals dependent on signaling by gradients of NGF.     

The duration of neurotrophic factor independence in early sensory neurons is matched to the time course of target field innervation.

To investigate how the onset of neurotrophic factor dependence in neurons is coordinated with the arrival of their axons in the target field, we have studied the survival of four populations of cranial sensory neurons whose axons reach their common central target field, the hindbrain, at different times. We show that neurons whose axons reach the hindbrain first survive for a short time in culture before responding to brain-derived neurotrophic factor (BDNF). Neurons whose axons reach the hindbrain later survive longer before responding to BDNF. These differences in survival, which arise prior to gangliogenesis, may play a role in coordinating trophic interactions for cranial sensory neurons.     

PTPsigma binds and dephosphorylates neurotrophin receptors and can suppress NGF-dependent neurite outgrowth from sensory neurons

Neurotrophin receptors of the Trk family play a vital role in the survival of developing neurons and the process of axonogenesis. The Trk family are receptor protein tyrosine kinases (RTKs) and their signalling in response to neurotrophins is critically dependent upon their ability to transphosphorylate and act as signalling centres for multiple adaptor proteins and distinct, downstream pathways. Such phosphotyrosine signalling also depends upon the appropriate counter-regulation by phosphatases. A large family of receptor-like protein tyrosine phosphatases (RPTPs) are also expressed in developing neurons and in this study we have examined the ability of the phosphatase PTPsigma to interact with and regulate Trk proteins in transfected HEK 293T cells. PTPsigma can bind differentially to Trk proteins, binding stably in complexes with TrkA and TrkC, but not TrkB. The transmembrane domains of PTPsigma and TrkA appear to be sufficient for the direct or indirect interaction between these two receptors. Furthermore, PTPsigma is shown to dephosphorylate all three Trk receptors and suppress their phosphorylation in the presence of neurotrophins. In addition, overexpression of PTPsigma in primary sensory neurons in culture inhibits neurite outgrowth without affecting the short-term survival of these neurons. PTPsigma can thus show differential complex formation with different Trk family members and in neurons can selectively target the neurite-forming signalling pathway driven by TrkA.     

Ectopic trkA expression mediates a NGF survival response in NGF- independent sensory neurons but not in parasympathetic neurons

We have investigated the role of trkA, the tyrosine kinase NGF receptor, in mediating the survival response of embryonic neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of brain-derived neurotrophic factor (BDNF) but not NGF, become NGF-responsive when microinjected with an expression vector containing trkA cDNA. In contrast, microinjection of ciliary neurotrophic factor (CNTF)-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF responsiveness by these neurons despite de novo expression of trkA mRNA and protein. The failure of trkA to result in an NGF-promoted survival response in ciliary neurons is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative RT/PCR and immunocytochemistry showed that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, namely NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.     

Ephrin-A5 inhibits growth of embryonic sensory neurons

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.     

Skin sensory innervation patterns in embryonic chick hindlimbs deprived of motoneurons

During normal development and following a variety of experimental manipulations (e.g., neural tube rotations, limb shifts), sensory neurons in the chick grow to their correct targets. L. Landmesser and M. G. Honig (1986, Dev. Biol. 118, 511-531) have suggested that sensory innervation may be precise, not because sensory neurons respond to limb-derived guidance cues, but because sensory neurons interact with motoneurons, which do respond to such cues. To test this hypothesis for skin sensory neurons, the ventral neural tube, including the motoneuron precursors, was removed from chick embryos prior to sensory axon outgrowth and the resulting patterns of dermatomes and axonal projections were mapped physiologically and anatomically. As reported previously, dorsal root ganglia (DRGs) and cutaneous nerves formed in their usual locations following the early removal of motoneurons, while most muscle nerves and the plexus region were reduced substantially (A. C. Taylor, 1944, J. Exp. Zool. 96, 159-185; L. Landmesser and M. G. Honig, 1986, Dev. Biol. 118, 511-531; G. J. Swanson and J. Lewis, 1986, J. Embryol. Exp. Morphol. 95, 37-52). The patterns of axonal projections and dermatomes were surprisingly, although not entirely, normal. In particular, cutaneous nerves in motoneuron-depleted embryos were derived from the same DRGs in approximately the same proportions as normal. Thus, while motoneurons may play a facilitative role in the development of the segmental pattern of skin sensory innervation, they do not appear to be essential.     

Calmodulin mediates brain-derived neurotrophic factor cell survival signaling upstream of Akt kinase in embryonic neocortical neurons

As a calcium-sensing protein, calmodulin acts as a transducer of the intracellular calcium signal for a variety of cellular responses. Although calcium is an important regulator of neuronal survival during development of the nervous system and is also implicated in the pathogenesis of neurodegenerative disorders, it is not known if calmodulin mediates these actions of calcium. To determine the role of calmodulin in regulating neuronal survival and death, we overexpressed calmodulin with mutations in all four Ca(2+)-binding sites (CaM(1-4)) or with disabled C-terminal Ca(2+)-binding sites (CaM(3,4)) in cultured neocortical neurons by adenoviral gene transfer. Long-term neuronal survival was decreased in neurons overexpressing CaM(1-4) and CaM(3,4), which could not be rescued by brain-derived neurotrophic factor (BDNF). The basal level of Akt kinase activation was decreased, and the ability of BDNF to activate Akt was completely abolished in neurons overexpressing CaM(1-4) or CaM(3,4). In contrast, BDNF-induced activation of p42/44 MAPKs was unaffected by calmodulin mutations. Treatment of neurons with calmodulin antagonists and a phosphatidylinositol 3-kinase inhibitor blocked the ability of BDNF to prevent neuronal death, whereas inhibitors of calcium/ calmodulin-dependent protein kinase II did not. Our findings demonstrate a pivotal role for calmodulin in survival signaling by BDNF in developing neocortical neurons by activating a transduction pathway involving phosphatidylinositol 3-kinase and Akt. In addition, our findings show that the C-terminal Ca(2+)-binding sites are critical for calmodulin-mediated cell survival signaling.     

Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell- substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Skin sensory innervation patterns in embryonic chick hindlimb following dorsal root ganglion reversals

During embryonic development skin sensory neurons in lumbosacral dorsal root ganglia (DRGs) establish their dermatomes and axonal projections in a precise, orderly fashion in the chick. To investigate mechanisms responsible for this specific outgrowth, the rostrocaudal order of DRGs T7-LS3 was reversed by rotating the corresponding segments of neural crest, either alone or together with the underlying neural tube in St.15-16 embryos. The resulting skin sensory innervation patterns, mapped physiologically or anatomically at St.29-40, differed between the two experimental groups. Following neural tube rotations DRGs tended to establish innervation patterns that were consonant with their original position in the embryo. Axons from these rotated DRGs generally projected into the appropriate pathways and innervated the appropriate region of skin. Neural crest rotations left the ventral neural tube (including the motor neuron precursors) largely intact. In this case rotated DRGs tended to establish innervation patterns in accordance with their new position in the embryo, almost as if no rotation had been made. These results cannot be explained solely by the inherent specificity of sensory neurons. Instead, the results are largely consistent with the suggestion (Honig et al., 1986; Landmesser and Honig, 1986) that motor axons can direct the outgrowth of sensory axons and thereby influence the establishment of sensory innervation patterns. Other mechanisms that may also affect the development of sensory innervation patterns are discussed.     

Subunit interactions inhibit the binding of beta nerve growth factor to receptors on embryonic chick sensory neurons

The binding of the beta nerve growth factor subunit in the 7 S nerve growth factor complex to the two nerve growth factor receptors on chick embryo dorsal root ganglion cells was investigated. Under conditions where the 7 S nerve growth factor complex is maximally stable (in the presence of excess alpha and gamma subunits and of 20 to 30 microM zinc ion), no binding to either receptor was detectable. The time course of the decrease in the binding of beta nerve growth factor to the receptors upon addition of alpha and gamma subunits and zinc ion paralleled the formation of the 7 S complex. Addition of alpha and gamma subunits and zinc ion to the bisdes-arginine118-nerve growth factor, which does not re-form the 7 S complex, failed to inhibit the binding of the derivative to either receptor. While the alpha subunit alone had no effect on beta nerve growth factor binding, the gamma subunit decreased its binding in proportion to the amount of complex formed between these two subunits, suggesting that the beta . gamma complex, like the 7 S complex, does not bind to nerve growth factor receptors.     

Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons.

An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10(-9)-10(-8)M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: high-affinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation in the peripheral nervous system.     

A low voltage-activated calcium conductance in embryonic chick sensory neurons.

Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the current's sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.     

Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.     

Patterned gene programs and target remodeling following axotomy at a major site for sensory innervation

The genetic mechanisms that a target uses to reestablish the connections of regenerating axons were explored using oligonucleotide microarrays and real-time PCR. In normal and denervated mouse vibrissa follicles, patterns of genetic regulation were assessed in adjacent targets that normally receive different types of sensory and autonomic innervation. We show that a target remodeling occurs following axotomy involving reduced hair growth, altered hair follicle integrity and remodeling of the extracellular matrix. Also, we found two orphan receptors putatively involved in hair growth. Our data further demonstrate region-specific regulation of genes putatively involved in target-axon interactions. Thus, this study shows for the first time that major target remodeling occurs following denervation and suggests putative functions for several novel genes.