Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal.

Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6-hydroxydopamine (6-OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves ß-adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of ß-adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

Neurotrophins regulate cholinergic synaptic transmission in cultured rat sympathetic neurons through a p75-dependent mechanism.

The sympathetic nervous system regulates many essential physiological systems, and its dysfunction is implicated in cardiovascular diseases. Mechanisms that control the strength of sympathetic output are therefore potential targets for the management of these disorders. Here we show that neurotrophins rapidly potentiate cholinergic transmission between cultured rat sympathetic neurons. We found that brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), acting at the p75 receptor, increased the amplitude of excitatory postsynaptic currents (EPSCs). We observed increased amplitude but not frequency of miniature synaptic currents after p75 activation, suggesting that p75 acts postsynaptically to modulate transmission at these synapses. This neurotrophic modulation enhances cholinergic EPSCs via sphingolipid signaling. Application of sphingolactone-24, an inhibitor of neutral sphingomyelinase, blocked the effect of BDNF, implicating a sphingolipid pathway. Furthermore, application of the p75-associated sphingolipid second messengers C(2)-ceramide and d-erythro-sphingosine restricted to the postsynaptic cell mimicked BDNF application. Postsynaptic blockade of ceramide production with fumonisin, a ceramide synthase inhibitor, blocked the effects of BDNF and d-erythro-sphingosine, implicating ceramide or ceramide phosphate as the active signal. Together these data suggest that neurotrophin signaling, which occurs in vivo via release from sympathetic neurons and target tissues such as the heart, acutely regulates the strength of the sympathetic postganglionic response to central cholinergic inputs. This pathway provides a potential mechanism for modulating the strength of sympathetic drive to target organs such as the heart and could play a role in the development of cardiovascular diseases.

Membrane depolarization induces p140trk and NGF responsiveness, but not p75LNGFR, in MAH cells.

Nerve growth factor (NGF) is required for the maturation and survival of sympathetic neurons, but the mechanisms controlling expression of the NGF receptor in developing neuroblasts have not been defined. MAH cells, an immortalized sympathoadrenal progenitor cell line, did not respond to NGF and expressed neither low-affinity NGF receptor (p75) nor p140trk messenger RNAs. Depolarizing concentrations of potassium chloride, but none of a variety of growth factors, induced expression of p140trk but not p75 messenger RNA. A functional response to NGF was acquired by MAH cells under these conditions, suggesting that expression of p75 is not essential for this response. Depolarization also permitted a relatively high proportion of MAH cells to develop and survive as neurons in fibroblast growth factor and NGF. These data establish a relation between electrical activity and neurotrophic factor responsiveness in developing neurons, which may operate in the functioning of the mature nervous system as well.

A v-myc-immortalized sympathoadrenal progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF.

Sympathetic neurons differentiate from a developmentally restricted progenitor cell in the neural crest-derived sympathoadrenal lineage. We have isolated these progenitors by fluorescence-activated cell sorting and immortalized them using a v-myc-containing retrovirus. The complement of antigenic markers expressed by these lines suggests that they have retained many of the properties of their normal counterparts. These lines initiate neuronal differentiation in response to basic FGF, but not to NGF, and do not contain NGF receptor mRNA. In NGF plus FGF, however, a small percentage of the cells differentiate to NGF-dependent postmitotic neurons. Furthermore, an induction of NGF receptor mRNA can be observed in response to FGF. Thus, the development of sympathetic neurons may involve a relay, in which FGF both initiates differentiation and induces the NGF receptor, which in turn controls further maturation and survival.

CNTF, FGF, and NGF collaborate to drive the terminal differentiation of MAH cells into postmitotic neurons.

The differentiation of neuronal cell progenitors depends on complex interactions between intrinsic cellular programs and environmental cues. Such interactions have recently been explored using an immortalized sympathoadrenal progenitor cell line, MAH. These studies have revealed that depolarizing conditions, in combination with exposure to FGF, can induce responsiveness to NGF. Here we report that CNTF, which utilizes an intracellular signaling pathway distinct from that of both FGF and NGF, can collaborate with FGF to promote efficiently the differentiation of MAH progenitor cells to a stage remarkably reminiscent of NGF-dependent, postmitotic sympathetic neurons. We also find that similar collaborative interactions can occur during transdifferentiation of normal cultured chromaffin cells into sympathetic neurons.

p75LNGFR regulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells.

We have examined NGF-induced signal transduction events and neuronal differentiation in MAH cells, a neuronal progenitor cell line, in which the expression of the two NGF receptors, p140trk (Trk) and p75LNGFR (p75), has been independently manipulated. Coexpression of a large molar excess of p75 substantially enhances the NGF-induced tyrosine autophosphorylation of Trk, compared with cells expressing Trk alone. MAH cells expressing both Trk and p75 stop dividing and acquire a mature neuronal morphology more rapidly and with greater efficiency than MAH cells expressing Trk alone. These biochemical and biological influences of p75 are not observed using a mutant form of NGF that binds Trk but not p75. These data provide evidence that p75 can modulate signal transduction through Trk in a neuronal progenitor cell context and that such modulation has functional consequences for the neuronal differentiation pathway induced by NGF.

Segregation of the classical transmitters norepinephrine and acetylcholine and the neuropeptide Y in sympathetic neurons: modulation by ciliary neurotrophic factor or prolonged growth in culture.

Recent evidence has demonstrated that cotransmission from mammalian neurons is not uniquely achieved by costorage and corelease of transmitters and cotransmitters from single varicosities, but also by the concurrent release of mediators segregated in separate synapses of individual neurons. An important question to be addressed is whether neurons show defined patterns of segregation or whether this is a plastic feature. We addressed this question by exploring the segregation pattern of the classical sympathetic transmitters norepinephrine (NE) and acetylcholine (ACh) and the cotransmitter neuropeptide Y (NPY) in sympathetic ganglionic neurons cocultured with cardiac myocytes. Using antibodies against NPY and the vesicular NE and ACh transporters VMAT2 and vesicular acetylcholine transporter (VAChT), we investigated the effect of ciliary neurotrophic factor (CNTF) or long (three weeks) culture periods on the segregation of VMAT2, VAChT, and NPY to separate varicosities. We found that although ganglionic neurons showed cell body coexpression of all the markers examined after three days, VMAT2 was segregated from VAChT in 43% of the VAChT-positive varicosities. In contrast, VMAT2 was only segregated from NPY in 16.3% of the NPY-positive varicosities. Cotransmitter segregation and VAChT expression was potentiated by both CNTF and longer times in culture. We also found two types of varicosities: one was smaller and located further from neuronal somata, and the other was larger, proximal to neuronal somata and had a higher level of segregation. These data demonstrate segregation of classical transmitters in sympathetic neurons and plasticity of neurotransmitter segregation. Finally, we discuss a possible functional correlate of segregation in sympathetic neurons.

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.

Postmigratory enteric and sympathetic neural precursors share common, developmentally regulated, responses to BMP2.

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.

Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors.

In vertebrates, the peripheral nervous system is embryologically derived from the neural crest. Although the earliest neural crest cells seem to be multipotent, the molecular mechanisms responsible for the restriction of these cells to different sublineages are not understood. We therefore searched for developmental control genes expressed in crest cells or their derivatives. One important class of regulatory molecules comprises proteins with common DNA-binding and dimerization domains, the basic helix-loop-helix (B-HLH) region. Members of this family include MyoD, a mammalian myogenic determination molecule, and proteins encoded by genes of the achaete-scute complex of Drosophila, which have an important role in neuronal determination. From a sympathetic neuronal precursor cell line derived from the neural crest we have now isolated two different mammalian genes that are homologous to genes of the achaete-scute complex. The sequence of the B-HLH-encoding region of these genes is more similar to that of the genes of the achaete-scute complex than it is to that of other, mammalian members of the B-HLH family. At least one of these genes is transiently expressed in the embryonic rat nervous system, is not detected in non-neuronal tissues or cell lines, and is induced by nerve growth factor in PC12 cells.

V-myc immortalization of early rat neural crest cells yields a clonal cell line which generates both glial and adrenergic progenitor cells.

We describe the isolation and characterization of an immortal cell line derived by infection of rat neural crest cells with a v-myc-containing replication-defective retrovirus. This clonal cell line, called NCM-1, contains a majority cell population with antigenic and morphologic properties that suggest it may represent a peripheral glial progenitor. In conditioned or in serum-free medium, these NGF receptor-positive cells differentiate to an elongated, bipolar morphology resembling that of primary Schwann cells. This morphologic differentiation is prevented by TGF-beta 1, which also acts as a mitogen for the cells. The NCM-1 line is also able to generate clonal derivatives which have extinguished expression of most or all glial markers. Once generated, such cells are stable and do not revert to the glial phenotype. At least some of these cells have acquired sympathoadrenal progenitor-like properties, as shown by their capacity to coexpress tyrosine hydroxylase (TH) and neurofilament (NF) in response to basic FGF and dexamethasone. These data imply that the NCM-1 line contains self-renewing cells with the potential to generate precursors in at least two of the sublineages that normally develop from the neural crest. This in turn suggests that the process of immortalization may preserve at least some of the developmental properties characteristic of multipotential neural crest cells. NCM-1 cells may prove useful for the study of neural crest cell lineage segregation, Schwann cell differentiation, and the mechanisms controlling the initial induction of TH and NF gene expression.

Nerve growth factor modulates synaptic transmission between sympathetic neurons and cardiac myocytes.

Regulation of heart rate by the sympathetic nervous system involves the release of norepinephrine (NE) from nerve terminals onto heart tissue, resulting in an elevation in beat rate. Nerve growth factor (NGF) is a neurotrophin produced by the heart that supports the survival and differentiation of sympathetic neurons. Here we report that NGF also functions as a modulator of sympathetic synaptic transmission. We determined the effect of NGF on the strength of synaptic transmission in co-cultures of neonatal rat cardiac myocytes and sympathetic neurons from the superior cervical ganglion (SCG). Synaptic transmission was assayed functionally, as an increase in the beat rate of a cardiac myocyte during stimulation of a connected neuron. Application of NGF produced a pronounced, reversible enhancement of synaptic strength. We found that TrkA, the receptor tyrosine kinase that mediates many NGF responses, is expressed primarily by neurons in these cultures, suggesting a presynaptic mechanism for the effects of NGF. A presynaptic model is further supported by the finding that NGF did not alter the response of myocytes to application of NE. In addition to the acute modulatory effects of NGF, we found that the concentration of NGF in the growth medium affects the level of synaptic transmission in cultures of sympathetic neurons and cardiac myocytes. These results indicate that in addition to its role as a survival factor, NGF plays both acute and long-term roles in the regulation of developing sympathetic synapses in the cardiac system.

Sympathetic neuroblasts undergo a developmental switch in trophic dependence.

Sympathetic neurons require NGF for survival, but it is not known when these cells first become dependent on neurotrophic factors. We have examined in vitro mitotically active sympathetic neuroblasts immuno-isolated from different embryonic stages, and have correlated this functional data with the expression of neurotrophin receptor mRNAs in vivo. Cells from E14.5 ganglia are supported by neurotrophin-3 (NT-3) in a serum-free medium, but not by NGF; NT-3 acts as a bona fide survival factor for these cells and not simply as a mitogen. By birth, sympathetic neurons are well-supported by NGF, whereas NT-3 supports survival only weakly and at very high doses. This change in neurotrophin-responsiveness is correlated with a reciprocal switch in the expression of trkC and trkA mRNAs by sympathetic neuroblasts in vivo. These data suggest that neurotrophic factors may control neuronal number at earlier stages of development than previously anticipated. They also suggest that the acquisition of NGF-dependence may occur, at least in part, through the loss of receptors for these interim survival factors.

DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer.

The MASH genes are vertebrate homologues of achaete-scute, genes required for neuronal determination in Drosophila. The sequence of MASH1 and MASH2 contains a basic helix-loop-helix (bHLH) motif that is present in other transcriptional regulators such as MyoD and E12. In the absence of an authentic target for the MASH proteins, we examined their DNA binding and transcriptional regulatory activity by using a binding site (the E box) from the muscle creatine kinase (MCK) gene, a target of MyoD. Like myogenic bHLH proteins, the MASH proteins form heterooligomers with E12 that bind the MCK E box with high affinity in vitro. Unexpectedly, however, MASH1 and MASH2 also activate transcription of both exogenous and endogenous MCK in transfected C3H/10T1/2 fibroblasts. However, they do not induce myogenesis. Myogenic activity is not exclusively a property of the MyoD basic region, as substitution of this domain fails to confer myogenic activity on MASH1. These data suggest that different bHLH proteins may activate overlapping but distinct sets of target genes in the same cell type.

Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons.

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle-containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors.

Developmental regulation of GDNF response and receptor expression in the enteric nervous system.

The development of the enteric nervous system is dependent upon the actions of glial cell line-derived neurotrophic factor (GDNF) on neural crest-derived precursor cells in the embryonic gut. GDNF treatment of cultured enteric precursor cells leads to an increase in the number of neurons that develop and/or survive. Here we demonstrate that, although GDNF promoted an increase in neuron number at all embryonic ages examined, there was a developmental shift from a mitogenic to a trophic response by the developing enteric neurons. The timing of this shift corresponded to developmental changes in gut expression of GFR alpha-1, a co-receptor in the GDNF-Ret signaling complex. GFR alpha-1 was broadly expressed in the gut at early developmental stages, at which times soluble GFR alpha-1 was released into the medium by cultured gut cells. At later times, GFR alpha-1 became restricted to neural crest-derived cells. GFR alpha-1 could participate in GDNF signaling when expressed in cis on the surface of enteric precursor cells, or as a soluble protein. The GDNF-mediated response was greater when cell surface, compared with soluble, GFR alpha-1 was present, with the maximal response seen the presence of both cis and trans forms of GFR alpha-1. In addition to contributing to GDNF signaling, cell-surface GFR alpha-1 modulated the specificity of interactions between GDNF and soluble GFR alphas. These experiments demonstrate that complex, developmentally regulated, signaling interactions contribute to the GDNF-dependent development of enteric neurons.

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130.

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.

Target cells promote the development and functional maturation of neurons derived from a sympathetic precursor cell line.

The role of target interactions in the development and functional maturation of peripheral neurons was investigated using an immortalized sympathetic precursor cell line. bMAH cells underwent neuronal differentiation in response to neurotrophic factors, but maintained an immature neuronal phenotype characterized by small cell bodies and continued cell division. Co-culture with cardiac myocytes, a target of sympathetic innervation, promoted the appearance of large-diameter postmitotic bMAH neurons. Analysis of bMAH maturation in the presence and absence of co-cultured myocytes indicated that myocyte-derived factors promoted the survival of maturing bMAH neurons prior to their acquisition of nerve growth factor dependence. Myocyte interactions also promoted the functional maturation of bMAH neurons, leading to an increase in the localization of synaptic vesicle proteins into neuritic varicosities and the acquisition of sympathetic-like intrinsic electrical properties. Like primary sympathetic neurons, mature bMAH neurons formed functional connections to cardiac myocytes as measured by evoked postsynaptic responses in connected myocytes. The effects of myocyte co-culture on developing bMAH neurons could be mimicked by myocyte conditioned medium, indicating that cardiac myocytes produce soluble factors that promote the appearance of mature neurons. These experiments indicate that targets of innervation play a role in directing the development and final maturation of peripheral neurons.