Neurofibromin negatively regulates neurotrophin signaling through p21ras in embryonic sensory neurons.

Embryonic sensory and sympathetic neurons that lack neurofibromin, the protein product of the neurofibromatosis type 1 (Nfl) gene, survive and extend neurites in the absence of neurotrophins. To determine whether neurofibromin negatively regulates neurotrophin signaling through its interaction with p21ras, we used Fab antibody fragments to block Ras function in DRG, trigeminal, nodose, and SCG neurons isolated from Nfl(-/-) and wild-type mouse embryos. We show that introduction of anti-Ras Fab fragments significantly reduces the ability of neurofibromin-deficient neurons to survive in the absence of neurotrophins. Moreover, addition of H-ras protein enhances the survival of Nfl(-/-), but not wild-type, DRG neurons. Our results are consistent with a major role for neurofibromin in modulating Trk signaling through p21ras during neuronal development.

Mouse tumor model for neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by increased incidence of benign and malignant tumors of neural crest origin. Mutations that activate the protooncogene ras, such as loss of Nf1, cooperate with inactivating mutations at the p53 tumor suppressor gene during malignant transformation. One hundred percent of mice harboring null Nf1 and p53 alleles in cis synergize to develop soft tissue sarcomas between 3 and 7 months of age. These sarcomas exhibit loss of heterozygosity at both gene loci and express phenotypic traits characteristic of neural crest derivatives and human NF1 malignancies.

Evidence of metabotropic glutamate receptor subtypes found on catfish horizontal and bipolar retinal neurons.

We have used electrophysiological, pharmacological and immunological techniques to determine which classes of metabotropic glutamate receptors exist on cone horizontal cells in the catfish retina. Patch-clamp recordings in acutely dissociated cone horizontal cells provide evidence that group I and III metabotropic glutamate receptors exist, and are linked to modulation of a voltage-gated calcium current. Group II metabotropic glutamate receptor agonists did not affect the calcium current. Immunocytochemical techniques were used to study the localization of metabotropic glutamate receptor subtypes found in the catfish retina. Antibodies raised against group I (metabotropic glutamate receptor 1alpha, metabotropic glutamate receptor 5), group II (metabotropic glutamate receptor 2/3) and group III (metabotropic glutamate receptor 6) metabotropic glutamate receptor subtypes were used to label acutely dissociated horizontal, bipolar and Müller cells. Results from immunostaining provide evidence that cone horizontal cells express group I (metabotropic glutamate receptor 1alpha, metabotropic glutamate receptor 5) and group III (metabotropic glutamate receptor 6), but not group II (metabotropic glutamate receptor 2/3) receptor subtypes, consistent with our electrophysiological results. Cone horizontal cells exposed to anti-metabotropic glutamate receptor 1alpha, 5 or 6 antibodies all demonstrated diffuse overall staining, with patches of dark immunostaining found on both dendritic processes and cell somata. In catfish bipolar cells, all four of the anti-metabotropic glutamate receptor antibodies stained the processes and cell bodies of bipolar cells homogeneously. There was no evidence for a group of bipolar cells that did not stain with the antimetabotropic glutamate receptor antibodies, although the densest immunostaining occurred when bipolar cells were incubated with the anti-metabotropic glutamate receptor 6 antibody. Müller cells did not show immunostaining against any anti-metabotropic glutamate receptor antibody. Our non-immune controls confirmed that immunostaining was specific for the antigen, and immunoblots were performed to demonstrate the specificity of the antibodies in catfish retina. These results support the hypothesis that group I and III metabotropic glutamate receptor subtypes are found on catfish horizontal cells, and group I, II and III metabotropic glutamate receptor subtypes are expressed on catfish bipolar cells. The metabotropic glutamate receptors on catfish cone horizontal cells act to modulate the voltage-gated sustained calcium current found on these cells.

Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway.

Neural crest cells of vertebrate embryos disperse on distinct pathways and produce different derivatives in specific embryonic locations. In the trunk of avian embryos, crest-derived cells that initially migrate on the lateral pathway, between epidermal ectoderm and somite, produce melanocytes but no neuronal derivatives. Although we found that melanocyte precursors are specified before they disperse on the lateral pathway, we also observed that a few crest-derived neuronal cells are briefly present on the same pathway. Here, we show that neuronal cells are removed by an episode of apoptosis. These observations suggest that localized environmental factor(s) affect the distribution of fate-restricted crest derivatives and function as a 'proof-reading mechanism' to remove 'ectopic' crest-derived cells.

Sympathetic neuron survival and proliferation are prolonged by loss of p53 and neurofibromin.

The proteins encoded by the p53 and Nf1 tumor suppressor genes are involved in cell signaling and regulation of proliferation during normal development and differentiation, as well as during tumor progression. To characterize the roles of these genes in the proliferation and survival of embryonic neurons, we have used dissociated cultures of sympathetic superior cervical ganglia (SCG) isolated from p53 and Nf1 single and compound-mutant mouse embryos. We have defined a temporal window for p53 involvement in sympathetic neuron survival and proliferation. Moreover, our results indicate that cooperativity between mutations in Nf1 and p53 prolongs SCG neuron proliferation and increases the incidence of neural tube defects in compound-mutant embryos.

Loss of neurofibromin results in neurotrophin-independent survival of embryonic sensory and sympathetic neurons.

Mutations at the neurofibromatosis 1 (NF1) locus in humans and mice result in abnormal growth of neural crest-derived cells, including melanocytes and Schwann cells. We have exploited a targeted disruption of the NF1 gene in mice to examine the role of neurofibromin in the acquisition of neurotrophin dependence in embryonic neurons. We show that both neural crest- and placode-derived sensory neurons isolated from NF1(-/-) embryos develop, extend neurites, and survive in the absence of neurotrophins, whereas their wild-type counterparts die rapidly unless nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) is added to the culture medium. Moreover, NF1 (-/-) sympathetic neurons survive for extended periods and acquire mature morphology in the presence of NGF-blocking antibodies. Our results are consistent with a model wherein neurofibromin acts as a negative regulator of neurotrophin-mediated signaling for survival of embryonic peripheral neurons.

Targeted mutation in the neurotrophin-3 gene results in loss of muscle sensory neurons.

Neurotrophin 3 (NT-3) is one of four related polypeptide growth factors that share structural and functional homology to nerve growth factor (NGF). NT-3 and its receptor, called neurotrophic tyrosine kinase receptor type 3 (Ntrk3; also called TrkC), are expressed early and throughout embryogenesis. We have inactivated the NT-3 gene in embryonic stem (ES) cells by homologous recombination. The mutated allele has been transmitted through the mouse germ line, and heterozygote intercrosses have yielded homozygous mutant newborn pups. The NT-3-deficient mutants fail to thrive and exhibit severe neurological dysfunction. Analysis of mutant embryos uncovers loss of Ntrk3/TrkC-expressing sensory neurons and abnormalities at early stages of sensory neuronal development. NT-3-deficient mice will permit further study of the role of this neurotrophin in neural development.

Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues.

The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.

Heterotopic transplantation of presumptive placodal ectoderm changes the fate of sensory neuron precursors.

The placode-derived cranial sensory neurons of the vestibular and nodose ganglia in avian embryos exhibit differences in neurite growth rate and the duration of neurotrophin-independent survival in vitro that arise prior to gangliogenesis and target contact (Davies, A. M. (1989) Nature 337, 553-555; Vogel, K. S. and Davies, A. M. (1991) Neuron 7, 819-830). To ascertain the state of commitment of presumptive placodal ectoderm to differentiate into neurons of the vestibular or nodose type, we performed heterotopic transplantation of labelled presumptive placodal ectoderm at E1.5 in the chicken embryo. We then assayed transplant-derived neurons for hindbrain innervation patterns, neurite growth and survival at E3.5. We show that presumptive placodal ectoderm is not determined to give rise to neurons of the vestibular or nodose phenotype at E1.5. Explantation of presumptive placodal ectoderm at E1.5 showed that this ectoderm is also not specified to differentiate into neurons at this stage. In addition, we demonstrate that non-neurogenic ectoderm from the trunk can give rise to nodose-type neurons when transplanted heterotopically to the nodose region.

Restriction of neurogenic ability during neural crest cell differentiation.

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells.

Development of trophic interactions in the vertebrate peripheral nervous system.

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of the trk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.

Neurotrophic factors promote the maturation of developing sensory neurons before they become dependent on these factors for survival.

We have studied the early development of chicken embryo sensory neurons in culture before they become dependent on neurotrophic factors for survival. During this period, they undergo a distinct change in morphology:initially they have small, spindle-shaped, phase-dark cell bodies, which become spherical and phase bright and extend long neurites. Although this maturational change occurs in isolated cells grown in chemically defined medium, it is accelerated by brain-derived neurotrophic factor (BDNF) or neurotrophin-3 and is retarded by antisense oligonucleotides that inhibit expression of the common, low affinity neurotrophic factor receptor (gp75NGFR) and by antisense BDNF oligonucleotides. We conclude that neurotrophic factors play a role in the earliest stages of sensory neuron development and suggest that they operate by an autocrine mechanism at this time.

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.

Developmental programmes of growth and survival in early sensory neurons.

In the developing vertebrate nervous system the survival of neurons becomes dependent on the supply of a neurotrophic factor from their targets when their axons reach these targets. To determine how the onset of neurotrophic factor dependency is coordinated with the arrival of axons in the target field, we have studied the growth and survival of four populations of cranial sensory neurons whose axons have markedly different distances to grow to reach their targets. Axonal growth rate both in vivo and in vitro is related to target distance; neurons with more distant targets grow faster. The onset trophic factor dependency in culture is also related to target distance; neurons with more distant targets survive longer before becoming trophic factor dependent. These data suggest that programmes of growth and survival in early neurons play an important role in coordinating the timing of trophic interactions in the developing nervous system.

Coordination of trophic interactions by separate developmental programs in sensory neurons and their target fields.

In the developing vertebrate nervous system the survival of sensory neurons becomes dependent on neurotrophic factors when their axons reach their target fields, and the synthesis of nerve growth factor (NGF) by target field cells commences with the arrival of the earliest axons. The timing of NGF synthesis and the onset of neurotrophic factor dependence are not, however, reliant on innervation. NGF synthesis occurs on time in developing target fields in which innervation is prevented, and sensory neurons cultured before innervating their targets become dependent on neurotrophic factors for survival after a certain length of time in culture. The length of time neurons survive in culture before becoming neurotrophic factor-dependent is related to the time they would normally contact their targets in vivo: populations of neurons that have nearby targets which are innervated early respond to neurotrophic factors before neurons that have more distant targets which are innervated later. The timing of target field innervation is governed not only by the distance axons have to grow but by the rate at which they grow. Axonal growth rate is also regulated in accordance with target distance: neurons with distant targets extend axons faster than neurons with nearby targets. In addition to reviewing evidence for separate developmental programs that control the timing of neurotrophic factor synthesis in the target field and the onset of neurotrophic factor dependence in early sensory neurons, we will consider the mechanisms that might play a role in regulating the survival of neurons during the phase of neurotrophic factor independence.

The sympathoadrenal lineage in avian embryos. I. Adrenal chromaffin cells lose neuronal traits during embryogenesis.

Cells of the sympathoadrenal lineage, including sympathetic neurons, adrenal chromaffin cells (pheochromocytes), and small intensely fluorescent (SIF) cells, arise from the neural crest. We have used antisera against catecholamine biosynthesis enzymes in conjunction with the monoclonal antibody A2B5 and an antiserum against the 160-kDa neurofilament (NF) protein, as markers of neuronal differentiation, to characterize the ontogeny of the sympathoadrenal lineage in quail embryos. The precursors of sympathetic neurons and pheochromocytes, present in the primary sympathetic chains, express neuronal traits and tyrosine hydroxylase (TH) early in development. The precursors that enter the developing adrenal gland from the primary sympathetic chain lose neuronal traits and later express the enzyme phenylethanolamine N-methyltransferase (PNMT). No TH+ cells differentiate in cultures of early (E7) embryonic adrenal glands after all A2B5+ cells have been immunoablated. When transplanted onto the neural crest migratory pathway, cells present in older (E13) embryonic adrenal glands can give rise to NF+ cells in the sympathetic ganglia. We conclude that both sympathetic neurons and pheochromocytes in avian embryos arise from a common bipotential precursor that initially expresses neuronal traits.

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells.

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.

Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes.

Neural crest cells obtained from explanted neural tubes take up, express, and retain exogenous DNA applied by the CaPO4 co-precipitation method during their differentiation into melanocytes. High efficiencies of gene transfer were obtained with both supercoiled DNA and intact phage particles; linear DNA or DNA from the phage yielded very low efficiencies. There is some evidence that transferred gene expression is differentiation dependent. The system should be useful for studies concerned with the analysis of cell developmental genes and their regulatory elements.

A subpopulation of cultured avian neural crest cells has transient neurogenic potential.

Neural crest cells of vertebrate embryos produce neurons, glia, pigment cells, and connective tissue in vivo and in vitro. To test the developmental potential of apparently undifferentiated crest cells, we have used the monoclonal antibody A2B5, which recognizes a cell surface glycolipid characteristic of neurons, to identify and immunoablate a subpopulation of cultured avian neural crest cells with a neuronal phenotype. Our results indicate that a limited neurogenic precursor subpopulation is present in cultures of avian neural crest cells and that the fate of this subpopulation can be influenced by environmental conditions arising when dispersal of neural crest cells is delayed.