Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation

In Xenopus, primary neurons differentiate early, in the deep layer of the neuroectoderm. In contrast, the neural precursors of the superficial layer continue to proliferate. We report that superficial layer precursors differ from deep layer precursors in that they are refractory to the neuronal-promoting activity of bHLH genes, dominant-negative X-Delta-1, FGF-8, or signals from the organizer. In this system, neuronal differentiation is guided by an early established, intrinsic, cell-autonomous difference in the competence of the precursor cells to differentiate. This difference may be controlled in part by ESR6e, a bHLH gene of the Enhancer-of-split family, which is expressed in the superficial layer of the late blastula and when expressed ectopically suppresses primary neurogenesis in the deep layer.     

Orthologs of key vertebrate neural genes are expressed during neurogenesis in the annelid Platynereis dumerilii

SUMMARY The molecular mechanisms underlying the formation and patterning of the nervous system are relatively poorly understood for lophotrochozoans (like annelids) as compared with ecdysozoans (especially Drosophila ) and deuterostomes (especially vertebrates). Therefore, we have undertaken a candidate gene approach to study aspects of neurogenesis in a polychaete annelid Platynereis dumerilii . We determined the spatiotemporal expression for Platynereis orthologs of four genes ( SoxB, Churchill, prospero / Prox , and SoxC) known to play key roles in vertebrate neurogenesis. During Platynereis development, SoxB is expressed in the neuroectoderm and its expression switches off when committed neural precursors are formed. Subsequently, Prox is expressed in all differentiating neural precursors in the central and peripheral nervous systems. Finally, SoxC and Churchill are transcribed in patterns consistent with their involvement in neural differentiation. The expression patterns of Platynereis SoxB and Prox closely resemble those in Drosophila and vertebrates—this suggests that orthologs of these genes play similar neurogenic roles in all bilaterians. Whereas Platynereis SoxC , like its vertebrate orthologs, plays a role in neural cell differentiation, related genes in Drosophila do not appear to be involved in neurogenesis. Finally, conversely to Churchill in Platynereis , vertebrate orthologs of this gene are expressed during neuroectoderm formation, but not later during nerve cell differentiation; in the insect lineage, homologs of these genes have been secondarily lost. In spite of such instances of functional divergence or loss, the present study shows conspicuous similarities in the genetic control of neurogenesis among bilaterians. These commonalities suggest that key features of the genetic program for neurogenesis are ancestral to bilaterians.     

Downregulation of CDC2 upon terminal differentiation of neurons.

The terminal differentiation of neurons occurs as precisely timed waves, with specific neuronal types differentiating in defined sequences. The precision of neuronal differentiation in the central nervous system offers an unusual opportunity to study terminal differentiation in vivo. The p34cdc2 kinase complex and the anti-oncogenes p53 and RB are central in the regulatory network that controls cell proliferation. We found high levels of expression of CDC2 mRNA and protein in proliferating neuronal precursor cells. The expression of both CDC2 and cyclin A was dramatically downregulated upon terminal differentiation of neurons in vivo and in a neuronal precursor cell line, ST15A. p53 mRNA expression was also downregulated but to a lesser extent; RB mRNA levels were unchanged during neuronal differentiation. Immunohistochemistry showed that p34cdc2 was expressed not only in the neuronal precursors of the cerebellar external granule layer but also in the early differentiating granule neurons. The expression of p34cdc2 in early neurons suggests a function for this enzyme in the events that occur soon after proliferation ceases. On the basis of the results reported here and other recent findings, we propose a model in which terminal differentiation is achieved by a switch in the neuronal precursors from p34cdc2-based proliferation to a differentiated state controlled by p34cdc2-related kinases.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

biparous:A Novel bHLH Gene Expressed in Neuronal and Glial Precursors inDrosophila

Genetic studies have uncovered many genes that are involved in the first steps of neuronal development inDrosophila.Less is known about the intermediate steps during which individual precursor cells follow either the neuronal pathway or the glial pathway. We report the identification of a novel bHLH gene,biparous,expressed in neuronal and glial precursors inDrosophila.Unlike most bHLH genes,biparousexpression continues to the final stages of neurogenesis in the embryo. Expression ofbiparousis not observed in end stage postmitotic neurons and precedes the expression ofrepo,a gene activated in later stages of glial differentiation. The bHLH domain is sufficiently different from previously described bHLH domains to imply a novel function.     

Differentiation of human adult skin-derived neuronal precursors into mature neurons

The isolation of autologous neuronal precursors from skin-derived precursor cells extracted from adult human skin would be a very efficient source of neurons for the treatment of various neurodegenerative diseases. The purpose of this study was to demonstrate that these neuronal precursors were able to differentiate into mature neurons. We isolated neuronal precursors from breast skin and expanded them in vitro for over ten passages. We showed that 48% of these cells were proliferating after the first passage, while this growth rate decreased after the second passage. We demonstrated that 70% of these cells were nestin-positive after the third passage, while only 17% were neurofilament M-positive after 7 days of differentiation. These neuronal precursors expressed betaIII tubulin, the dendritic marker MAP2 and the presynaptic marker synaptophysin after 7 days of in vitro maturation. They also expressed the postsynaptic marker PSD95 and the late neuronal markers NeuN and neurofilament H after 21 days of differentiation, demonstrating they became terminally differentiated neurons. These markers were still expressed after 50 days of culture. The generation of autologous neurons from an accessible adult human source opens many potential therapeutic applications and has a great potential for the development of experimental studies on normal human neurons.     

cato encodes a basic helix-loop-helix transcription factor implicated in the correct differentiation of Drosophila sense organs

In Drosophila neurogenesis, proneural genes encode bHLH proteins that are required for neural precursor selection. But many vertebrate homologues are expressed later and are postulated to have multiple roles during neurogenesis. We have isolated a new Drosophila gene, cato, which encodes a protein with a bHLH domain that is closely related to that of the proneural protein Atonal. cato expression is restricted to the developing PNS, where it is expressed in between the stages of precursor selection and terminal differentiation (and therefore later than the proneural genes). We present evidence from loss-of-function and misexpression experiments that cato is involved in sensory neurone morphology. Moreover, in prospero mutants, in which axon and dendrite outgrowth is defective, cato is strongly derepressed in the developing CNS.     

L-myc and N-myc influence lineage determination in the central nervous system.

The N-myc and the L-myc proto-oncogenes are expressed during embryonal development mainly in the developing brain. Studies of their expression in single neuroepithelial cells revealed that neural precursors not yet committed to the glial or the neuronal lineage expressed both genes, but after lineage commitment they expressed either N-myc or L-myc. Moreover, enforced expression of L-myc in the neural precursor cell line 2.3D caused neuronal differentiation, while the expression of N-myc promoted glial differentiation. These results indicate that L-myc and N-myc play critical roles in lineage determination for the central nervous system.     

The expression and function of MTG/ETO family proteins during neurogenesis

The proneural basic helix-loop-helix (bHLH) proteins promote neurogenesis by inducing changes in gene expression required for neuronal differentiation. Here we characterize one aspect of this differentiation program by analyzing a small family of putative corepressors encoded by MTG genes. We show that MTG genes are expressed sequentially during neurogenesis as cells undergo neuronal differentiation in both the chick spinal cord and in the Xenopus primary nervous system. Using in ovo electroporation, we show that misexpressing wild-type forms of MTG proteins in the developing chick spinal cord does not detectably alter neuronal differentiation. By contrast, the number of differentiated neurons is markedly reduced when a putative dominant-negative mutant of the MTG proteins is expressed in neural precursors in a manner that can be rescued by wild-type MTGR1. Together, these results suggest that MTG family members act downstream of proneural proteins, presumably as corepressors, to promote neuronal differentiation.     

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.     

Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2

Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. We show here that lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons, as the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain.