Early Stages Of Neuronal Differentiation Research Articles

Gi-coupled prostanoid receptors are the likely targets for COX-1-generated prostanoids in rat pheochromocytoma (PC12) cells

Cyclooxygenase-1 (COX-1) behaves as a delayed response gene in rat pheochromocytoma (PC12) cells exposed to nerve growth factor (NGF). To investigate the possible targets for COX-1 generated prostanoids in the early stages of neuronal differentiation, we have examined the expression of prostanoid receptors by PC12 cells using functional assays. Prostanoid receptor-specific agonists failed to activate adenylyl cyclase in undifferentiated and NGF-treated PC12 cells; neither did they stimulate phospholipase C activity. EP3 receptor agonists and PGF 2α were the only active ligands, able to inhibit forskolin-stimulated adenylyl cyclase activity. PC12 cells expressed EP3 and FP receptor mRNA, but only the responses to EP3 receptor agonists were inhibited by the EP3 receptor antagonist ONO-AE3-240. The functional role of NGF-stimulated COX-1 remains to be determined since we found no strong evidence of a role for EP3 receptors in the morphological changes induced by NGF during the early stages of differentiation of PC12 cells.

AUF1 and Hu proteins in the developing rat brain: Implication in the proliferation and differentiation of neural progenitors

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU-rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal-specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real-time RT-PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down-regulation of AUF1 from P1, whereas HuB was strongly up-regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP-43 was not detected in HuD-transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.

Signaling pathways of the early differentiation of neural stem cells by neurotrophin-3

Neurotrophin-3 (NT-3) is well known to play an important role in facilitating neuronal survival and differentiation during development. However, the mechanisms by which neurotrophin-3 promotes prolonged Akt/MAPK signaling at an early stage are not well understood. Here, we report that NT-3 works at an early stage of neuronal differentiation in mouse neural stem cells (NSCs). After treatment with NT-3 for 12 h, more NSCs differentiated into neurons than did untreated cells. These findings demonstrated that stimulation with NT-3 causes NSCs to differentiate into neurons through a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the phosphorylated extracellular signal-regulated kinase (ERK) pathway. In addition, treatment with NT-3 induced neurite outgrowth by specific phosphorylation of p38 MAPK, which was accompanied by neuronal differentiation. Taken together, these results suggest that NT-3, along with the Trk C receptors in NSCs, might lead to the survival and neuronal differentiation of NSCs via two distinct downstream signaling pathways at an early stage of neuronal differentiation.

Distinct expression pattern of microtubule-associated protein/microtubule affinity-regulating kinase 4 in differentiated neurons

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate tau protein and related microtubule-associated proteins (MAPs), and their role in the establishment of cell polarity in different contexts. Recent papers have indicated that microtubule affinity-regulating kinase 4 ( MARK4) is a gene that is finely regulated at transcriptional level and expressed in two spliced isoforms called MARK4L and MARK4S. We here describe the characterization of the mouse orthologue of the human MARK4 gene. Interestingly, MARK4S is predominantly expressed in the brain, whereas MARK4L shows lower transcript levels in this organ. Using MARK4 antibodies specific for each isoform, we found that both isoforms have an identical expression pattern in the mouse CNS, and are present in a number of neuronal populations. We also found that human microtubule affinity-regulating kinase 4S (hMARK4S), whose expression is not detectable in human neural progenitor cells (HNPCs) and NTera2 (NT2) cells, is up-regulated in both cell systems from the very early stages of neuronal differentiation. This indicates that neuronal commitment is marked by MARK4S up-regulation. In conclusion, this study provides the first direct evidence suggesting that MARK4 is a neuron-specific marker in the CNS, and the up-regulation of MARK4S during neuronal differentiation suggests that it plays a specialized role in neurons.

Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition

Revealing the mechanisms for neuronal somatic diversification remains a central challenge for understanding individual differences in brain organization and function. Here we show that an engineered human LINE-1 (for long interspersed nuclear element-1; also known as L1) element can retrotranspose in neuronal precursors derived from rat hippocampus neural stem cells. The resulting retrotransposition events can alter the expression of neuronal genes, which, in turn, can influence neuronal cell fate in vitro. We further show that retrotransposition of a human L1 in transgenic mice results in neuronal somatic mosaicism. The molecular mechanism of action is probably mediated through Sox2, because a decrease in Sox2 expression during the early stages of neuronal differentiation is correlated with increases in both L1 transcription and retrotransposition. Our data therefore indicate that neuronal genomes might not be static, but some might be mosaic because of de novo L1 retrotransposition events.

Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis

The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast-like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6-suppressed P19 (P19[anti-Sox6]) cells by transfection of antisense-Sox6 cDNA. Most of the P19[anti-Sox6] cells showed no neurites and were not stained by the anti-MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA-dependent apoptosis by P19[anti-Sox6] cells: RA-treated P19[anti-Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase-3-like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

The effect of experimental epilepsy induced by penicillin administration during pregnancy on nestin expression in the immature rat cerebellum. A light, electron microscopic, and immunohistochemical study.

Recent knowledge regarding the effect of epileptic seizures in pregnant women on newborns was limited and, therefore, it was difficult to suggest the proper clinical guidelines and to take precautions against it. Studies evaluating the morphological effects of epileptic seizure during pregnancy on newborns in various experimental models are valuable. Therefore, the current study was designed to investigate the morphological changes in the cerebellum of newborn pups of rats subjected to experimental epilepsy during pregnancy. Swiss Albino rats were divided into three groups (six animals in each). In the first group (experimental group) an acute grand mal epileptic seizure was induced by 400 IU penicillin-G administration into their intrahippocampal CA3 region with a stereotaxic device during the 13th day of their pregnancy. The second group (intrahippocampal saline-injected sham group) and the third group (untreated animals) were the control groups. On the 1st neonatal day, pups were perfused with intracardiac fixative solution under anesthesia, and newborn cerebellums were dissected surgically for light and electron microscopic studies. In an immunohistochemical study using Rat-401 monoclonal antibody and peroxidase, the intermediate filament nestin was detected in the developing cerebellar tissue. Histologically, normal migration and cerebellar maturation were determined in the newborn rat cerebellum in the control and sham-operated groups. It was observed that the morphological structure of the cerebellar cortex in the experimental group was compromised in the early embryonal period. In contrast to the control and sham groups, it was found that nestin (+) cell density was increased in the experimental epilepsy group. It has been concluded that epileptic convulsions during embryonic life may cause early neurogenesis and delayed maturation, which explains the harmful effects of epileptic grand mal seizures, hypoxia, and obstetric trauma to the embryo at the early stage of neuronal differentiation. However, further studies are necessary to investigate epileptic pregnant phenomena and to characterize the possible relationship between epilepsy and congenital malformations as well as mental retardation.

Implication of Trip15/CSN2 in early stage of neuronal differentiation of P19 embryonal carcinoma cells

Trip15/CSN2 is a transcriptional corepressor/a component of COP9 signalosome (CSN) and participates in various signaling pathways. However, participation of Trip15/CSN2 in neural differentiation is still obscure. Here, we show that Trip15/CSN2 plays a critical role in neuronal differentiation. The expression of Trip15/CSN2 mRNA was induced at an early stage of neuronal differentiation in the retinoic acid (RA)-treated P19 cells, but not in the triiodothyronine (T3)-primed cardiac muscular cell differentiation. The expression of Trip15/CSN2 mRNA in the rat brain was detected at E14 and the protein was localized in the nuclei of neonatal rat CNS neurons. Enforced expression of sense rat Trip15/CSN2 mRNA caused the downregulation of Oct-3/4 mRNA expression and was sufficient to convert P19 cells into neurons, but not glial cells, only after the aggregation without RA. In the presence of RA, exogenous expression of the sense mRNA caused the intense and rapid induction of neurogenic Brn-2 and Mash-1 mRNA expressions accompanying the strong downregulation of Oct-3/4 mRNA expression, and stimulated both neuronal and glial cell differentiations of P19 cells. In contrast, enforced expression of the antisense mRNA suppressed the commitment of RA-treated aggregation form of P19 cells to neuronal lineage. These data strongly suggest that Trip15/CSN2 could implicate in the commitment of multipotent embryonal carcinoma (EC) cells to neuronal fate through the downregulation of Oct-3/4 which suppresses neurogenic genes. Moreover, in addition to Trip15/CSN2, RA-regulated other factor(s) may be required for glial cell differentiation.

Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo

Unlike mammals, adult urodele amphibians can regenerate their spinal cord and associated ganglia, but the molecular mechanisms controlling regeneration are not fully understood. We have recently shown that expression of FGF2, a member of the fibroblast growth factor family, is induced in the progenitor cells of the regenerating spinal cord and appears to play a role in their proliferation and possibly in their differentiation. In order to investigate which receptor(s) may mediate FGF2 signaling and their role in regeneration, we have studied expression of the four fibroblast growth factor receptors, FGFR1, FGFR2, FGFR3 and FGFR4, and of the spliced variants, sFGFR and KGFR, in the regenerating spinal cord of the adult urodele, Pleurodeles waltl, following tail amputation. We show that all FGFRs are expressed in normal and regenerating spinal cord, with the exception of the spliced variants that are expressed only in non-neural tissues of the tail. FGFR1 and 4 show the more interesting spatio-temporal patterns of expression. They are not detectable in the ependymal cells of normal cords, from which neural progenitors for regeneration are believed to originate, though they are expressed in some mature neurons. During regeneration, significant up-regulation of FGFR1 precedes that of FGFR4 in the ependymal tube from which the new cord will form. FGFR4 is highly expressed in these cells at later stages of regeneration, when neuronal differentiation is becoming apparent, and like FGFR1 is also expressed in some newborn neurons. In addition to the known form of FGFR1, the antibody against this receptor reacts also with a non-phosphorylated protein that appears to be present only during regeneration, and might represent a yet undescribed variant of the receptor. Altogether this study shows that fibroblast growth factor signaling is finely modulated during tail and spinal cord regeneration, and points to FGFR1 and FGFR4 as key players in this process, suggesting that FGFR1 is primarily associated with proliferation of progenitor cells and FGFR4 with early stages of neuronal differentiation.

Neuronal T-type α1H Calcium Channels Induce Neuritogenesis and Expression of High-Voltage-Activated Calcium Channels in the NG108–15 Cell Line

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108–15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the α1H T-channel subunit. Furthermore, we describe that inhibition of α1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

Neuronal T-type alpha 1H calcium channels induce neuritogenesis and expression of high-voltage-activated calcium channels in the NG108-15 cell line.

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108-15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the alpha1H T-channel subunit. Furthermore, we describe that inhibition of alpha1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

Conditionally Immortalized Clonal Cell Lines from the Mouse Olfactory Placode Differentiate into Olfactory Receptor Neurons

To test extracellular signals that direct the development of the olfactory system, we have generated clonal temperature-sensitive cell lines that represent distinct cellular lineages derived from the E10 mouse olfactory placode. Two of these lines, OP6 and OP27, express (at the permissive temperature), a transcriptional profile representing intermediate-late developmental stages in the olfactory receptor neuron (ORN) lineage. At the nonpermissive temperature, both OP6 and OP27 cells can be induced by all-trans retinoic acid to differentiate into a population of mature bipolar ORN-like cells. In response to retinoic acid, differentiated OP6 and OP27 down-regulate neuron-specific transcription factors required for early stages of neuronal differentiation, and shift active components of the neurotrophin signaling cascade (Trk receptors) into a kinase inactive state. When morphologically mature, OP6 and OP27 express the mature ORN chemosensory signaling components, olfactory G-protein (Golf), Type III adenylate cyclase (ACIII), OCNC1, and the olfactory marker protein (OMP). OP27 expresses one odorant receptor, OR 27-3. OP6 expresses two very closely related receptors, OR 6–13 and OR 6–8. Voltage-gated sodium and potassium channels resembling those recorded from primary cultures of ORNs can also be recorded from a subset of differentiated OP6 cells.

Temporal Appearance of the Presynaptic Cytomatrix Protein Bassoon during Synaptogenesis

Bassoon is a 420-kDa presynaptic cytomatrix protein potentially involved in the structural organization of neurotransmitter release sites. In this study, we have investigated a possible role for Bassoon in synaptogenesis and in defining synaptic vesicle recycling sites. We find that it is expressed at early stages of neuronal differentiation in which it is selectively sorted into axons. As synaptogenesis begins, Bassoon clusters appear along dendritic profiles simultaneously with synaptotagmin I, sites of synaptic vesicle recycling, and the acquisition of functional excitatory and inhibitory synapses. A role for Bassoon in the assembly of excitatory and inhibitory synapses is supported by the colocalization of Bassoon clusters with clusters of GKAP and AMPA receptors as well as GABAA receptors. These data indicate that the recruitment of Bassoon is an early step in the formation of synaptic junctions.

Neuronal Localization of Presenilin-1 and Association with Amyloid Plaques and Neurofibrillary Tangles in Alzheimer’s Disease

Mutations in the presenilin-1 (PS1) gene is a cause of early- onset familial Alzheimer's disease (AD). Endogenous PS1 is associated with the endoplasmic reticulum in the cell body of undifferentiated SH-SY5Y neuroblastoma cells. At early stages of neuronal differentiation in rat hippocampal culture, PS1 appears in all neuritic processes and in growth cones. In mature differentiated neurons, PS1 is concentrated in the somatodendritic compartment but is also present at lower levels in axons. A similar localization of PS1 is observed in vivo in neurons of the adult human cerebral cortex. In sporadic AD, PS1 appears in the dystrophic neurites of mature amyloid plaques and co-localizes with a subset of intraneuronal neurofibrillary tangles (NFTs). About 30% of hippocampal NFTs are labeled with a highly specific antibody to the PS1 C-terminal loop domain but not with an antibody to the PS1 N terminus. This observation is consistent with a potential association of the PS1 C-terminal fragment with NFTs, because PS1 is constitutively cleaved to N- and C-terminal fragments in neurons. These results suggest that PS1 is highly expressed and broadly distributed during early stages of neuronal differentiation, consistent with a role for PS1 in neuronal differentiation. Furthermore, the co-localization of PS1 with NFTs and plaque dystrophic neurites implicates a role for PS1 in the diverse pathological manifestations of AD.

Differentiation to an NGF-Dependent State and Apoptosis Following NGF Removal Both Occur Asynchronously in Cultures of PC12 Cells

Long-term timelapse videomicroscopy was used to investigate the relationships and transitions between mitosis, differentiation, and apoptosis in cultures of NGF-differentiated PC12 cells. After 4 days in NGF, cultures were at an early stage of neuronal differentiation. Removal of NGF led to an appreciable increase in apoptosis with no effect on the relatively high mitotic rate. After 7 days in NGF, cells were more neuronal; NGF withdrawal again resulted in no change in the low mitotic rate but an even greater increase in apoptosis, eventually leading to considerable net loss of cells. After 10 days, cells were terminally differentiated; removal of NGF did not affect the negligible mitotic rate but induced a dramatic increase in apoptosis resulting in death of most of the cells. Apoptosis in the fraction of cells that had become NGF-dependent followed a similar timecourse and was characterized by the same morphology at all three differentiation states. Thus, acquisition of NGF-dependence in PC12 cultures seemed to be the result of a steadily increasing percentage of cells that had each undergone a relatively rapid transition to a postmitotic, NGF-sensitive state. These studies were also helpful for elucidating the timing of apoptosis. Onset of apoptosis was markedly asynchronous within a culture, but the active, blebbing phase, once initiated, always lasted about 45 min, regardless of differentiation state or time spent without NGF. Thus, the active phase might represent a conserved sequence of events that every cell must ultimately undergo before apoptotic death.

Changes in arachidonic acid levels and formation and in lipid synthesis in the human neuroblastoma SK-N-BE during retinoic acid-induced differentiation.

We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.

Developmental regulation of MAP2 variants during neuronal differentiation in vitro

Microtubule-associated protein 2 (MAP2) is a major component of the neuronal cytoskeleton and is known to promote the assembly and stabilization of microtubules, functions which have important implications in neuronal differentiation. MAP2 consists of high molecular weight (HMW) proteins MAP2a, MAP2b and a low molecular weight (LMW) isoform MAP2c which are produced from a single gene by alternative splicing. In this study, we describe the expression of the various MAP2 mRNA isotypes and protein isoforms during the development of rat cerebellar granule cell neurons over a 21-day period in vitro. In situ hybridization was used to detect MAP2 mRNA isotypes which corresponded to HMW- and LMW-MAP2 proteins. The distribution of MAP2 mRNAs in the developing P7 cerebellar cortex was related to the different stages of granule neuron development in situ. During early stages of neuronal differentiation in vitro, high levels of MAP2c mRNA were observed which gradually decreased as development progressed. Throughout the period studied, MAP2ab mRNA concentrations remained low although a small transient rise was noted during the first 14 days in vitro (div). The profile of MAP2 protein variants showed further developmental regulation. The expression of the LMW-MAP2c isoform closely mirrored that of its mRNA whilst HMW-MAP2b protein concentrations rose during the first 10 div and were maintained in older cultures. HMW-MAP2a appeared after 4 div and gradually increased throughout the remainder of the study. Clearly, the outline of HMW-MAP2 protein did not relate to its encoding mRNA and such disparity may be due to the operation of different transcriptional and/or posttranslational mechanisms. Immunocytochemical analyses of MAP2 variants provided further information concerning their localization during neurite outgrowth. These results describe the developmental regulation of MAP2 mRNA and protein variants and that the profile of their expression relates to the formation of processes during the differentiation of granule neurons in vitro.

Expression of Rb, E2F1, cdc2, and D, and B cyclins in developing spinal cord

The spatial and temporal distribution of transcripts for the tumor suppressor gene Rb, transcription factor E2F1, cdc2 kinase, cyclins D1, D2, B1 and B2 during neurogenesis of the spinal cord was determined by in situ hybridization. The Rb and E2F1 transcripts were detectable in proliferating and differentiating cells. By contrast, cdc2, cyclins D1, B1 and B2 are expressed in the ventricular zone where proliferating cells are localized. Cyclin D2 mRNA was detectable only in the marginal zone of the developing neural tube. Electrophoretic mobility shift analyses demonstrated a changing pattern of DNA/protein complexes that bind to E2F binding site. These observations suggest that Rb and E2F1 may be involved in the early stages of neuronal differentiation in addition to the cell cycle regulation.

Participation of the mitochondrial genome in the differentiation of neuroblastoma cells.

Using clonal cell lines isolated from murine neuroblastoma C1300, we investigated the mitochondrial changes related to neuronal differentiation and, more generally, the role played by the mitochondrion in this phenomenon. By different approaches (measurement of the mitochondrial mass, immunoquantification of specific mitochondrial proteins, or incorporation of Rhodamine 123), the differentiation of the inducible clone, N1E-115, was found associated with an important increase of the cellular content in mitochondria. This increase could be observed with differentiating N1E-115 cells maintained in suspension, i.e. under conditions where neurite outgrowth is prevented but other early stages of (biochemical) differentiation continue to occur. That these mitochondrial changes are likely to be correlated with these stages of neuronal differentiation, rather than with simple progression to the postmitotic stage, stems from comparative experiments with clone N1A-103, a neuroblastoma cell line variant that becomes postmitotic after induction but fails to differentiate and shows no modification in its cellular content in mitochondria. In accordance with these observations, chloramphenicol prevents differentiation when added together with the inducer. This effect is probably related to the inhibition of mitochondrial translation rather than to modification of the bioenergetic needs because oligomycine, a potent inhibitor of the mitochondrial ATP synthetase, shows no effect on neurogenesis. As a working hypothesis and in keeping with independently published models, we postulate that products resulting from mitochondrial translation could be involved in the organization of the cytoskeleton or of certain membrane components whose rearrangements should be the prerequisite or the correlates to early stages of neuronal differentiation.

Mammalian neuronal differentiation: early expression of a neuronal phenotype from mouse neural crest cells in a chemically defined culture medium

We show that mouse neural crest cells cultured in a serum-deprived chemically defined medium on appropriate culture substrata can be induced to express a neuronal phenotype. The uncommitted neural crest cells express a mesenchymal intermediate filament protein such as vimentin, but not the usual neuronal markers such as receptor sites for tetanus toxin or neurofilaments. In the chemically defined medium, receptor sites for tetanus toxin or neurofilaments can be characterized after a few hours in culture. Furthermore, these cells acquire tetrodotoxin-sensitive voltage-dependent Na+ channels and can generate action potentials. Such an in vitro system should allow us to analyze and manipulate early stages of neuronal differentiation in a mammalian embryo, at a level so far restricted to lower vertebrate embryos.