Neural Crest-derived Sensory Neurons Research Articles

Elp1 is required for development of visceral sensory peripheral and central circuitry.

ABSTRACTCardiovascular instability and a blunted respiratory drive in hypoxic conditions are hallmark features of the genetic sensory and autonomic neuropathy, familial dysautonomia (FD). FD results from a mutation in the gene ELP1, the encoded protein of which is a scaffolding subunit of the six-subunit Elongator complex. In mice, we and others have shown that Elp1 is essential for the normal development of neural crest-derived dorsal root ganglia sensory neurons. Whether Elp1 is also required for development of ectodermal placode-derived visceral sensory receptors, which are required for normal baroreception and chemosensory responses, has not been investigated. Using mouse models for FD, we here show that the entire circuitry underlying baroreception and chemoreception is impaired due to a requirement for Elp1 in the visceral sensory neuron ganglia, as well as for normal peripheral target innervation, and in their central nervous system synaptic partners in the medulla. Thus, Elp1 is required in both placode- and neural crest-derived sensory neurons, and its reduction aborts the normal development of neuronal circuitry essential for autonomic homeostasis and interoception. This article has an associated First Person interview with the first author of the paper.

Developmental origin of peripheral ciliary band neurons in the sea urchin embryo

In the sea urchin larva, most neurons lie within an ectodermal region called the ciliary band. Our understanding of the mechanisms of specification and patterning of these peripheral ciliary band neurons is incomplete. Here, we first examine the gene regulatory landscape from which this population of neural progenitors arise in the neuroectoderm. We show that ciliary band neural progenitors first appear in a bilaterally symmetric pattern on the lateral edges of chordin expression in the neuroectoderm. Later in development, these progenitors appear in a salt-and-pepper pattern in the ciliary band where they express soxC, and prox, which are markers of neural specification, and begin to express synaptotagminB, a marker of differentiated neurons. We show that the ciliary band expresses the acid sensing ion channel gene asicl, which suggests that ciliary band neurons control the larva’s ability to discern touch sensitivity. Using a chemical inhibitor of MAPK signaling, we show that this signaling pathway is required for proper specification and patterning of ciliary band neurons. Using live imaging, we show that these neural progenitors undergo small distance migrations in the embryo. We then show that the normal swimming behavior of the larvae is compromised if the neurogenesis pathway is perturbed. The developmental sequence of ciliary band neurons is very similar to that of neural crest-derived sensory neurons in vertebrates and may provide insights into the evolution of sensory neurons in deuterostomes.

A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal.

Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons.

Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos

Microtubules are the primary cytoskeletal constituent of extending neurites. We used antisense morpholinos to knock down expression of neuron-specific Class III beta tubulin in the right half of the neural tube of chicken embryos in ovo. There was a significant (p < 0.01) reduction in the number of Class III beta tubulin immunostained interneurons 24 h following electroporation of the morpholinos when compared with the contralateral side of the neural tube. However, neural crest-derived sensory neurons labeled with the fluorescently tagged morpholinos developed distinct processes. Moreover, there was no significant difference in the number of interneurons labeled on either side of the neural tube with a second marker of developing neurons, anti-microtubule associated protein (MAP) 1b. Neural tubes were also excised and dissociated following antisense or control morpholino electroporation. The resulting neurons were cultured for 48 h and immunostained with anti-Class III beta tubulin and anti-MAP 1b. Neurons that had taken up the antisense morpholino had significantly shorter neurites (p < 0.01) than neurons from the same neural tubes that did not; they also had significantly shorter neurites (p < 0.05) than labeled neurons from neural tubes electroporated with a control morpholino. Thus, normal expression of Class III beta tubulin may not be necessary for neurogenesis in the early avian spinal cord in situ, but is required for neurite outgrowth in vitro.

A direct role for Sox10 in specification of neural crest-derived sensory neurons

sox10 is necessary for development of neural and pigment cell derivatives of the neural crest (NC). However, whereas a direct role for Sox10 activity has been established in pigment and glial lineages, this is more controversial in NC-derived sensory neurons of the dorsal root ganglia (DRGs). We proposed that sox10 functioned in specification of sensory neurons, whereas others suggested that sensory neuronal defects were merely secondary to absence of glia. Here we provide evidence that in zebrafish, early DRG sensory neuron survival is independent of differentiated glia. Critically, we demonstrate that Sox10 is expressed transiently in the sensory neuron lineage, and specifies sensory neuron precursors by regulating the proneural gene neurogenin1. Consistent with this, we have isolated a novel sox10 mutant that lacks glia and yet displays a neurogenic DRG phenotype. In conjunction with previous findings, these data establish the generality of our model of Sox10 function in NC fate specification.

MKlf7, a potential transcriptional regulator of TrkA nerve growth factor receptor expression in sensory and sympathetic neurons.

Development of the nervous system relies on stringent regulation of genes that are crucial to this process. TrkA, the receptor for nerve growth factor (NGF), is tightly regulated during embryonic development and is essential for the survival and differentiation of neural crest-derived sensory and sympathetic neurons. We have previously identified a mouse TrkA enhancer and have characterized several cis regulatory elements that are important for appropriate TrkA expression in vivo. We now report the cloning of a novel gene encoding a Kruppel-like factor from a mouse dorsal root ganglion expression library. This Kruppel-like factor, named mKlf7, binds specifically to an Ikaros core binding element that is crucial for in vivo TrkA enhancer function. Using in situ hybridization, we demonstrate that mKlf7 is coexpressed with TrkA in sensory and sympathetic neurons during embryogenesis and in adulthood. These data are consistent with the idea that mKlf7 may directly regulate TrkA gene expression in the peripheral nervous system.

NGF expression in the developing rat brain: effects of maternal separation

A number of studies have shown that mothering style in rodents can produce neuroendocrine, neurochemical and behavioural changes in the adult, although the basic mechanisms initiating this cascade of events still need to be investigated. Long term changes in neuronal function might be due to alterations in the expression of neurotrophins which have been shown to promote neuronal survival, differentiation and function during development, such as Nerve Growth Factor (NGF). NGF is essential for proper development of sympathetic and neural crest-derived sensory neurons of the peripheral nervous system as well as of central cholinergic neurons. In previous studies, using a maternal separation paradigm, we have shown that NGF expression is increased in the dentate gyrus and the hilus of the hippocampus as a result of brief (45 min) maternal separations. In the present study neonatal rats were separated for longer periods of time (up to 3 h) and at different ages during development (9 and 16 days postnatally). Results indicate that the effects of maternal separation on NGF expression are stronger with longer separations and are not restricted to the hippocampal region but can be seen also in other brain areas. Overall these results indicate that external factors, such as the presence/absence of the mother, can modify neurotrophic factor’s availability in the brain, thus indicating NGF as a potential player in environmentally-mediated brain plasticity during development.

Molecular characterization of the TrkA/NGF receptor minimal enhancer reveals regulation by multiple cis elements to drive embryonic neuron expression.

Neural development relies on stringent regulation of key genes that mediate specialized function. TrkA is primarily expressed in neural crest-derived sensory and sympathetic neurons where it transmits critical survival information. We have identified a 457 base pair sequence upstream of the murine first TrkA coding exon that is conserved in human and in chick, and is sufficient for expression in the correct cells with appropriate timing. Mutation analysis of consensus transcription factor binding domains within the minimal enhancer reveals a complex positive regulation that includes sites required for global expression and sites that are specifically required for DRG, trigeminal or sympathetic expression. These results provide a foundation for identification of the transcriptional machinery that specifies neurotrophin receptor expression.

Loss of brain-derived neurotrophic factor-dependent neural crest-derived sensory neurons in neurotrophin-4 mutant mice.

Peripheral ganglion neurons confer sensory information including touch, pain, temperature, and proprioception. Sensory modality is linked to specific neurotrophin (NTF) requirements. NT-3 supports survival of neurons that differentiate primarily into proprioceptors whereas nerve growth factor and brain-derived neurotrophic factor (BDNF) support subpopulations that transmit nociception and mechanoreception, respectively. We examined sensory neurons of gene-targeted mouse mutants at the NT-4, BDNF, NT-3, and TrkA loci. We show that NT-4 functions early in gangliogenesis, upstream of BDNF. In the absence of NT-4 function, BDNF-dependent, TrkB-expressing neurons fail to appear. The results are consistent with the model that precursor cells intended to become BDNF-dependent mechanoreceptors instead differentiate into NT-3-dependent proprioceptive neurons.

RXR? gene is expressed by discrete cell columns within the alar plate of the brainstem of the chicken embryo

With in situ hybridization assays, we mapped the distribution of retinoid X receptor gamma (RXR gamma) gene transcripts in the central nervous system of the chicken embryo. Previous studies have demonstrated the presence of RXR gamma transcripts in migrating neural crest and in neural crest derivatives throughout the peripheral nervous system, implicating RXR gamma as an early pan-neural crest marker (Rowe et al. 1991. Development 111:771-778), and in the retina (Hoover et al. 1998. J Comp Neurol 391:204-213). Here we report the presence of RXR gamma transcripts in discrete regions of the developing neural tube, within the hindbrain, the cerebellar plate, the optic tectum, and the diencephalon. At stage 10, when migrating neural crest expresses RXR gamma transcripts, we detect no transcripts in the neural tube. By stage 13, RXR gamma transcripts accumulate to detectable levels along the midline of the posterior optic tectum, where the neural crest-derived sensory neurons of the mesencephalic trigeminal nucleus are located. By stage 15, RXR gamma transcripts also appear in an intermittent longitudinal cell column within the mantle zone of the alar plate of the hindbrain, eventually extending into the cerebellar plate rostrally and into the cervical spinal cord caudally, with a gap at about rhombomere 3. By stage 19, transcripts appear in a discrete population of cells within the diencephalon. Expression in these cell populations continues until at least stage 22.5, when many neuron populations have been generated in the hindbrain. The localization of the RXR gamma-positive cells to the mantle zone suggests that they are postmitotic and are probably neurons. Their specific alar locations indicate that they reside within sensory columns and potential downstream targets, evidently corresponding to some of the central components of the trigeminal system.

P21 Ras and Phosphatidylinositol-3 Kinase Are Required for Survival of Wild-Type and NF1 Mutant Sensory Neurons

Nerve growth factor (NGF) is a required differentiation and survival factor for sympathetic and a majority of neural crest-derived sensory neurons in the developing vertebrate peripheral nervous system. Although much is known about the function of NGF, the intracellular signaling cascade that it uses continues to be a subject of intense study. p21 ras signaling is considered necessary for sensory neuron survival. How additional intermediates downstream or in parallel may function has not been fully understood yet. Two intracellular signaling cascades, extra cellular regulated kinase (erk) and phosphatidylinositol-3 (PI 3) kinase, transduce NGF signaling in the pheochromocytoma cell line PC12. To elucidate the role these cascades play in survival and differentiation, we used a combination of recombinant adenoviruses and chemical inhibitors to perturb these pathways in sensory neurons from wild-type mice and mice deficient for neurofibromin in which the survival and differentiation pathway is constitutively active. We demonstrate that ras activity is both necessary and sufficient for the survival of embryonic sensory neurons. Downstream of ras, however, the erk cascade is neither required nor sufficient for neuron survival or overall differentiation. Instead, the activity of PI 3 kinase is necessary for the survival of the wild-type and neurofibromin-deficient neurons. Therefore, we conclude that in sensory neurons, NGF acts via a signaling pathway, which includes both ras and PI 3 kinase.

The time course of nerve growth factor content in different neuropsychiatric diseases--a unifying hypothesis.

Nerve growth factor (NGF) is the prototype of related neurotrophic proteins, the so-called neurotrophins. NGF is essential for proper development of sympathetic and neural crest-derived sensory neurons of the peripheral nervous system (PNS) as well as of the neurons in the cholinergic basal forebrain (CBF). In the mature peripheral and central nervous system (CNS) NGF is also biologically active; NGF facilitates neuronal plasticity and regulates synaptic transmission and connectivity. Besides this well established neurotrophic function, recent findings suggest a role of NGF in neuroimmune and stress-associated processes, which NGF imparts not only as the classical "target-derived messenger", that is retrogradely transported within NGF-sensitive neurons, but also as para- and autocrine cytokine modulating the function of non-neuronal cells. Since neurotrophins are produced in very small amounts in vivo, NGF-sensitive cells have to compete for the limited NGF even under physiological conditions, so that normally only less than 10% of NGF receptors (NGFR) are saturated with their endogenous ligand. Consequently, it is feasable that minute changes in NGF concentrations can influence neuronal function in an extensive way. Hence, one plausible pathomechanism of disease(s) may be that a deficiency in NGF leads to malfunction of NGF-sensitive neurons. The change in NGF concentrations in the course of several diseases, namely during alcoholic and diabetic neuropathy as well as in Alzheimer's disease (AD) and several lesion-models of the CBF, indicates that fluctuations of endogenous NGF concentrations in PNS and CNS follow a distinctive pattern. An initial deficit of NGF at the onset of pathological processes is typically followed by its temporary elevation, during which some neuronal deficits may be partially ameliorated. However, if the disease progresses a decrease of NGF is typically observed, which appears to be a "normalization" of formerly elevated NGF concentrations. In our hypothesis we postulate that after acute or chronic injuries NGF is up-regulated as an intrinsic attempt to regenerate NGF-sensitive neurons. After long-term exposure to noxious processes, however, this compensatory increase of NGF cannot be maintained and eventually breaks down. The extent of such a compensatory up-regulation may depend on age and condition of NGF-sensitive neurons as well as on the type of lesion (acute or chronic). Furthermore, we also postulate that an exceeding level of NGF or its chronic elevation could even be detrimental to neuronal functioning under certain conditions. Thus, endogenous NGF has the capacity to modulate and even to compensate different kinds of harmful processes and in this way it may reinstate the homeostatic equilibrium. In our view, it seems to be a more appropriate approach to regard NGF changes as independent of classical constructs of neuropsychiatric diseases. Perhaps our understanding of NGF may even model for a new approach to the aetiology of multifactorial neuropsychiatric disorders.

Chapter 5 Developmental changes in the neurotrophic factor survival requirements of peripheral nervous system neurons

Publisher Summary This chapter shows that neurons are generated in excess in the developing vertebrate nervous system, and those that are superfluous to requirement are eliminated in a phase of cell death that occurs shortly after they innervate their targets. The proposal that neuronal survival is dependent on and regulated by substances synthesized in limiting amounts by their target fields has been substantiated by work on nerve growth factor (NGF). The tissues innervated by NGF-dependent neurons contain trace quantities of NGF in proportion to their innervation density and these neurons are eliminated in development by function-blocking anti-NGF antibodies or by null mutations in the NGF or NGF receptor tyrosine kinase (trkA) genes. This chapter discusses the neurotrophic factor switching in trigeminal ganglion neurons. Several populations of neural crest-derived sensory neurons change their neurotrophin requirements during the development. The majority of neurons of the jugular ganglion and the neural crest-derived part of the trigeminal ganglion of chicken embryos are supported in culture by BDNF, NT3, and NGF early in their development, and subsequently lose responsiveness to BDNF and NT3, but remain dependent on NGF. This chapter discusses these other populations of sensory neurons and ends with a look at the sympathetic neurons.

Screen for mutations affecting development of Zebrafish neural crest.

The neural crest provides a useful model to learn how cell fate diversification is regulated during vertebrate development. Our approach is to isolate zebrafish mutations in which the development of neural crest derivatives is disrupted, in order to learn about the underlying genetic mechanisms. We describe a screen in which parthenogenetic diploid embryos are examined both for visible phenotypes and for cellular defects in neural crest-derived sensory neurons recognized immunohistochemically. We present preliminary results from this screen and briefly describe a few representative mutations. We also discuss the general utility of our strategy and comment on the future directions of this approach.

Screen for mutations affecting development of zebrafish neural crest

The neural crest provides a useful model to learn how cell fate diversification is regulated during vertebrate development. Our approach is to isolate zebrafish mutations in which the development of neural crest derivatives is disrupted, in order to learn about the underlying genetic mechanisms. We describe a screen in which parthenogenetic diploid embryos are examined both for visible phenotypes and for cellular defects in neural crest-derived sensory neurons recognized immunohistochemically. We present preliminary results from this screen and briefly describe a few representative mutations. We also discuss the general utility of our strategy and comment on the future directions of this approach. © 1996 Wiley-Liss, Inc.

Nerve growth factor (NGF) in cerebrospinal fluid (CSF) from patients with various neurological disorders.

It has been recently shown that NGF is not only involved in the survival and development of sympathetic and neural crest-derived sensory neurons, but also in some mechanisms of the immune system. For this reason, we studied the content of NGF in CSF samples from patients with diseases in which neuroimmunological mechanisms seem to be involved (multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer disease, chronic relapsing polyradiculoneuritis, Guillain-Barré syndrome, and tumors of the nervous system), as well as from a number of normal control subjects. We setup an ELISA aimed at the beta subunit of NGF, obtaining good validation tests and a detection limit of 28 pg beta NGF per ml. None of the samples was found to contain detectable levels of NGF and, when a concentration method for sample enrichment was used, only one patient was NGF-positive. This suggests that NGF is probably not involved in the neuroimmunological mechanisms underlying some inflammatory and degenerative diseases of the nervous system.

Trophic protection of motor neurons: clinical potential in motor neuron diseases.

Recent advances in understanding the physiologic role of nerve growth factor (NGF), obtained both from tissue culture and efficacy studies in animals, have suggested that neurotrophic factors may have clinical potential in the treatment of neurodegenerative diseases or nerve trauma [12,21]. First characterized as a target-derived survival factor for developing sympathetic and sensory neurons, it is now clear that NGF plays an important role in the maintenance and regeneration of mature peripheral neurons. Prompted by in vitro findings, it was established in the mid-1980's that intracerebroventricular infusions of NGF are capable of rescuing basal forebrain cholinergic neurons from axotomy-induced cell death produced by fimbria-fornix lesions. Given that degeneration of cholinergic neurons is a major contributing factor in the loss of cognitive function in Alzheimer's disease, there has been a great deal of interest in exploring the therapeutic potential of NGF in this disease [16]. The highly restricted specificity of NGF for sympathetic neurons, sub-populations of neural crest-derived sensory neurons and striatal and basal forebrain cholinergic neurons has for almost two decades spurred the search for other neurotrophic factors with specificities directed to the many classes of neurons which do not respond to NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related effects of nerve growth factor on the morphology of embryonic sensory neurons in vitro.

Studies of neonatal and adult mammals have shown that neuronal morphology is regulated in part by the availability of target-derived neurotrophic factor. To test whether the same is true for embryonic neurons, which are dependent on target-derived neurotrophic factors for survival, we grew neural crest-derived sensory neurons from the trigeminal ganglion of avian embryos of different ages in vitro in different concentrations of nerve growth factor (NGF) and measured the number of branch points and total length of the resulting arborizations. Although the size and complexity of arborizations increased with embryonic age up to embryonic day (E)14, neuronal morphology for embryos younger than E14 was unaffected by the concentration of NGF in the culture medium. However, beginning at E14, the stage at which trigeminal neurons start to lose their absolute requirement for NGF for survival, the neurons had significantly more branch points and larger arborizations in higher concentrations of NGF. Thus, it appears that the extent of neurite outgrowth in young embryos is independent of neurotrophic factor concentration; each neuron that receives enough neurotrophic factor to survive elaborates approximately the same size arbor. As trigeminal neurons mature and become less dependent on neurotrophic factor for survival, they acquire the ability to respond to neurotrophic factor with increased neurite growth and branching, as in neonates and adults.

Receptors for Nerve Growth Factor

This chapter discusses the two receptors for nerve growth factor (NGF)—namely, (1) the low-affinity NGF receptor and (2) the product of the proto-oncogene trk . It also reviews the way they were originally identified and the structural and functional features of the two receptors. NGF interacts with two receptor glycoproteins in responsive cells, p75 NGFR and p140 Prototrk . Both receptors are expressed on NGF-responsive cells, including sympathetic and neural crest-derived sensory neurons, cholinergic neurons of the basal forebrain, and PC12 cells, a widely used cell culture model for studying the mechanism of action of NGF. Two different receptors interact with NGF, leading to cell survival and cell differentiation and maintenance of neurons. The finding that NGF binding to trk can induce cell proliferation suggests new roles for neurotrophic factors. The product of the proto-oncogene trk is the primary signaling NGF receptor, the function of which is to increase the tyrosine phosphorylation of cellular proteins. Sensory deficits in mutant mice missing the P75 NGFR gene suggest that this receptor has an essential role in neuronal development.

A “calcium set-point hypothesis” of neuronal dependence on neurotrophic factor

In this commentary, we discuss evidence suggesting that cytoplasmic free calcium concentration determines neurotrophic factor dependence. Developing sympathetic and neural crest-derived sensory neurons require nerve growth factor (NGF) for survival both in vivo and in vitro. Chronic depolarization of these cells in culture causes a modest sustained elevation of cytoplasmic calcium concentration and promotes their survival in the absence of NGF. The amount of calcium increase caused by depolarization is closely correlated with the ability of the cells to survive in NGF-free medium. At an optimal calcium concentration, that we refer to as a “set point,” survival is equivalent to that of cells grown in the presence of NGF.