Uwe Ernsberger

Proliferation and differentiation of embryonic chick sympathetic neurons: Effects of ciliary neurotrophic factor

At early developmental stages (embryonic day 7, E7), chick paravertebral sympathetic ganglia contain a cell population that divides in culture while expressing various neuronal properties. In an attempt to identify factors that control neuronal proliferation, we found that ciliary neurotrophic factor (CNTF) specifically inhibits the proliferation of those cells expressing neuronal markers. In addition, CNTF affects the differentiation of sympathetic ganglion cells by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). After 1 day in culture, tyrosine hydroxylase immunoreactivity (TH-IR) was expressed by about 86% of the cells whereas VIP-IR was virtually absent. In the presence of CNTF, 50%-60% of the cells expressed VIP-IR after 4 days in culture; however, none of the cells expressed VIP-IR in the absence of CNTF. These results, and the demonstration of cells that express both VIP and TH-IR, indicate that VIP is induced in cells that initially express tyrosine hydroxylase. The findings suggest a potential role for CNTF as a factor affecting the proliferation and differentiation of developing sympathetic neurons.

Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels “tunes” heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the generation and survival of various DRG neuron classes, in particular proprioceptors. Its importance for peripheral projections and central connectivity of proprioceptors demonstrates the significance of NT signalling for integrating responsive neurons in neural networks. The molecular targets of NT3 signalling in proprioceptor differentiation remain to be characterized. In sympathetic ganglia, NGF signalling regulates dendritic development and axonal projections. Its role in the specification of other neuronal properties is less well analysed. In vitro analysis suggests the involvement of NT signalling in the choice between the noradrenergic and cholinergic transmitter phenotype, in the expression of various classes of ion channels and for target connectivity. In vivo analysis is required to show the degree to which NT signalling regulates these sympathetic neuron properties in developing embryos and postnatally.

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.

Neuronal precursor cells in chick dorsal root ganglia: Differentiation and survival in vitro

Neuronal precursor cells present in dorsal root ganglia (DRG) during early development have been previously shown to differentiate in vitro to neurons, as characterized by morphology, cell surface antigens, and electrophysiological properties (H. Rohrer, S. Henke-Fahle, T. El-Sharkawy, H. D. Lux, and H. Thoenen, 1985, Embo J. 4, 1709-1714). In the present study the conditions necessary for the initial differentiation and long-term survival of these cells were established, and the neurotransmitter phenotype of the newly differentiated neurons was analyzed. Neuronal precursor cells isolated from chick DRG at Embryonic Day 6 (E6) were found to require the presence of a polyornithine substrate coated with either laminin or fibronectin for initial neurite production and long-term survival. Neurons were unable to develop on polyornithine alone or on polyornithine coated with BSA. The survival and neurite outgrowth from neuronal precursor cells was not affected by the presence of nerve growth factor (NGF) during the first 9 hr in culture. NGF also had no effect on the proportion of cells expressing the neuron-specific Q211 antigen. However, after this initial differentiation period the neurons did require the presence of a survival factor. The neurons could be maintained for at least 6 days in culture both in the presence of NGF and in the presence of brain-derived neurotrophic factor (BDNF). At saturating concentrations of both survival factors no additive effects could be observed, indicating a complete overlap of NGF- and BDNF-responsiveness. Although the same proportion of cells survived with either NGF or BDNF during the first 3 days in culture, survival decreased in the presence of BDNF but not in the presence of NGF during the following 3 days in culture. The loss of BDNF responsiveness in vitro was also observed in vivo. After 6 days in culture about 70% of the neurons expressed substance P immunoreactivity, and approximately the same proportion was positive for myelin-associated glycoprotein immunoreactivity. The neurons did not express properties of adrenergic neurons such as tyrosine hydroxylase immunoreactivity or norepinephrine uptake. These findings indicate that the neuronal precursor cells from E6 DRG acquire the same characteristics in vitro as in their normal in vivo environment.

THE EXPRESSION OF TYROSINE HYDROXYLASE AND THE TRANSCRIPTION FACTORS CPHOX-2 AND CASH-1 : EVIDENCE FOR DISTINCT INDUCTIVE STEPS IN THE DIFFERENTIATION OF CHICK SYMPATHETIC PRECURSOR CELLS

The adrenergic differentiation of sympathetic neurons is controlled by complex interactions with the embryonic environment. To provide a basis for the experimental analysis of these interactions, the expression of the adrenergic marker enzyme tyrosine hydroxylase (TH) was analyzed by immunohistochemistry and in situ hybridization in sympathetic ganglia and the adrenal medulla of chick embryos. In parallel, the developmental expression of the transcription factors cPhox-2 and Cash-1 was analyzed by in situ hybridization. TH protein was first detectable during the third day of development (stage 19) in cells of the primary sympathetic strands. A few hours earlier (stage 18), TH mRNA could be found by in situ hybridization. At the very same time and location, mRNA for the transcription factor cPhox-2 was first observed. In contrast, mRNA for the transcription factor Cash-1 was detected much earlier, at stage 15, dorsal to the aorta where the primary sympathetic ganglia form. High TH mRNA levels are maintained during later embryonic development (stage 35) in both sympathetic ganglia and adrenal chromaffin cells. In contrast, cPhox-2 and Cash-1 mRNA are selectively reduced in chromaffin cells and sympathetic ganglia, respectively. The results show that TH and cPhox-2 are early markers expressed in sympathetic ganglia. Their coordinated expression points towards an inductive event possibly occurring close to the aorta and leading to the expression of an adrenergic phenotype. Cash-1 is detected significantly earlier, suggesting that its expression is induced by a separate event.

The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation.

Cholinergic properties in chick sympathetic neurons are detectable early during development of paravertebral ganglia and mature after target contact. The cholinergic marker choline acetyltransferase (ChAT) is first detectable at embryonic day 6 and its expression partly overlaps with that of the noradrenergic marker tyrosine hydroxylase (TH). At late embryonic stages, when sympathetic neurons have established target contact, ganglia consist of two major neuronal populations, TH-positive noradrenergic neurons and cholinergic neurons that at this stage express vasoactive intestinal peptide (VIP) in addition to ChAT. The maturation of sympathetic neurons is paralleled by changes in their response to the neurokine ciliary neurotrophic factor (CNTF). These findings suggest that expression of neurotransmitter properties is controlled differentially before and during target innervation.

The expression of dopamine β-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympathetic neurons: evidence for common regulation during noradrenergic induction and diverging regulation later in development

During differentiation of sympathetic neurons in chick embryos, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) mRNAs become detectable during the same developmental period and are both induced by BMP 4. Later during sympathetic ganglion development, DBH is detectable in TH-positive and -negative cells. Moreover, BMPs reduce DBH mRNA in cultures of sympathetic neurons while leaving TH unaffected. The data provide evidence for a common regulation of TH and DBH early during sympathetic neuron differentiation and indicate that BMPs promote their initial expression but not the maintenance during later development. The time course of Phox2a and 2b expression suggests an evolutionary conserved role in noradrenergic induction. In addition, Phox2a, Phox2b, and c-ret may be involved in the differentiation of cholinergic sympathetic neurons.

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.

The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c‐ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss‐of‐function mutation of the c‐ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate‐limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT‐positive cells are affected by the c‐ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c‐ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.