Properties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression

doi:10.1006/mcne.1998.0712

Molecular and Cellular Neuroscience

Volume 12, Issues 4–5, November 1998, Pages 281-299

https://doi.org/10.1006/mcne.1998.0712 Get rights and content

Abstract

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3β, which inhibits NeuroD functionin vivo,blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

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Embryos injected with 0.5 ng of Ngnr1-GR mRNA were treated with dexamethasone at the early (stage 10.5) or late (stage 12.5) gastrula stages and analyzed for gene expression at the neurula (stage 15) or tailbud (stage 27) stages. Activation of Ngnr1-GR at the gastrula stages resulted in a reduction of the NC-specific gene Snail2, and a dramatic expansion of N-Tub and Pak3 expression domain at the neurula stage, converting the entire ectoderm into primary neuron progenitors (Table 4; Fig. 7A), as previously reported (Ma et al., 1996; Olson et al., 1998; Perron et al., 1999). For both genes the lateral expansion was more pronounced for a treatment with dexamethasone at stage 10.5 (Fig. 7A).
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This suggests a two-step model of radial migration, with a first step in which early postmitotic neurons exit the VZ/SVZ neuroepithelium and migrate into the IZ, which requires phosphorylation of Ngn2Y241, leading to transient inhibition of RhoA activity through transcription of RhoGAPs and also expression of Doublecortin and other regulators of neuronal migration (Mattar et al., 2004; Bai et al., 2003); and a second step controlled by Rac1/Cdc42 activity (Kawauchi et al., 2003) in which neurons migrate through from the IZ into the CP (reviewed in Gupta et al., 2002). Several studies have already illustrated the importance of protein-protein interactions and posttranslational modifications in mediating some of the biological functions of bHLH transcription factors (Lee and Pfaff, 2003; Moore et al., 2002; Olson et al., 1998; Sun et al., 2001; Talikka et al., 2002; Vojtek et al., 2003; reviewed in Puri and Sartorelli, 2000). Our results show that the DNA binding-independent function of Ngn2 in the specification of the neuronal migration properties and dendritic morphology of cortical neurons is mediated by phosphorylation of tyrosine 241.
The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2^Y241F mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.
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Neural stem cells proliferate and maintain multipotency when cultured in the presence of FGF2, but subsequent lineage commitment by the cells is nevertheless influenced by the exposure to FGF2. Here we show that FGF2 effects on neural stem cells are mediated, in part, by β-catenin. Conversely, the effects of β-catenin in neural stem cells depend in part upon whether there is concurrent fibroblast growth factor (FGF) signaling. FGF2 increases β-catenin signaling through several different mechanisms including increased expression of β-catenin mRNA, increased nuclear translocation of β-catenin, increased phosphorylation of GSK-3β, and tyrosine phosphorylation of β-catenin. Overexpression of β-catenin in the presence of FGF2 helps to maintain neural progenitor cells in a proliferative state. However, overexpression of β-catenin in the absence of FGF2 enhances neuronal differentiation. Further, chromatin immunoprecipitation (ChIP) assays demonstrate that both β-catenin and Lef1 bind directly to the neurogenin promoter, and luciferase reporter assays demonstrate that β-catenin is directly involved in the regulation of neurogenin 1 and possibly other proneural genes when neural stem cells are cultured in the presence of FGF2. We suggest that the balance between the mitogenic effects and the proneural effects of β-catenin is determined by the presence of FGF signaling.
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2003, Developmental Biology
Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19^INK4d activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19^INK4d in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19^INK4d expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development.
The secreted glycoprotein noelin-1 promotes neurogenesis in Xenopus
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Neurogenesis in Xenopus neural ectoderm involves multiple gene families, including basic helix-loop-helix transcription factors, which initiate and control primary neurogenesis. Equally important, though less well understood, are the downstream effectors of the activity of these transcription factors. We have investigated the role of a candidate downstream effector, Noelin-1, during Xenopus development. Noelin-1 is a secreted glycoprotein that likely forms large multiunit complexes. In avians, overexpression of Noelin-1 causes prolonged and excessive neural crest migration. Our studies in Xenopus reveal that this gene, while highly conserved in sequence, has a divergent function in primary neurogenesis. XenopusNoelin-1 is expressed mainly by postmitotic neurogenic tissues in the developing central and peripheral nervous systems, first appearing after neural tube closure. Its expression is upregulated in ectopic locations upon overexpression of the neurogenic genes X-ngnr-1 and XNeuroD. Noelin-1 expression in animal caps induces expression of neural markers XBrn-3d and XNeuroD, and co-expression of secreted Noelin-1 with noggin amplifies noggin-induced expression of XBrn-3d and XNeuroD. Furthermore, in animal caps neuralized by expression of noggin, co-expression of Noelin-1 causes expression of neuronal differentiation markers several stages before neurogenesis normally occurs in this tissue. Finally, only secreted forms of the protein can activate sensory marker expression, while all forms of the protein can induce early neurogenesis. This suggests that the cellular localization of Noelin-1 may be important to its function. Thus, Noelin-1 represents a novel secreted factor involved in neurogenesis.
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During primary neurogenesis in Xenopus, a cascade of helix–loop–helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.

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¹: To whom correspondence and reprint requests should be addressed at present address: Department of Neurology, Beth-Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston MA 02115. Fax: (617) 667-0810. E-mail:[email protected]

²: Present address: Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA 92037.

³: Present address: Cell Press, 1050 Massachusetts Avenue, MA 02138.

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Regular ArticleProperties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression

Abstract

Kidney Int.

Neuron

Curr. Biol.

Dev. Biol.

Cell

Cell

Adv. Genet.

Neuron

Dev. Biol.

Cell

Curr. Opin. Neurobiol.

Biochem. Biophys. Res. Commun.

Curr. Opin. Neurobiol.

Neuron

Cell

Mech. Dev.

Neuron

Mol. Cell. Neurosci.

Cell

Cell

Biochem. Biophys. Res. Commun.

Notch signaling

Science

Developmental changes in the inward current of the action potential of Rohon-Beard neurones

J. Physiol. (London)

Differentiation of voltage-gated potassium current and modulation of excitability in cultured amphibian spinal neurones

J. Physiol. (London)

The appearance and development of chemosensitivity in Rohon-Beard neurones of the Xenopus spinal cord

J. Physiol. (London)

The appearance and development of neurotransmitter sensitivity in Xenopus embryonic spinal neurones in vitro

J. Physiol. (London)

The activity of neurogenin1 is controlled by local cues in the zebrafish embryo

Development

Genetic mechanisms of early neurogenesis in Drosophila melanogaster

Mol. Neurobiol.

Green fluorescent protein as a marker for gene expression

Science

Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta

Nature

Neural induction and neurogenesis in amphibian embryos

Perspect Dev. Neurobiol.

Sensitivity of proneural genes to lateral inhibition affects the pattern of primary neurons in Xenopus embryos

Development

Xotch, the Xenopus homolog of Drosophila notch

Science

Role of glycogen synthase kinase 3β as a negative regulator of dorsoventral axis formation inXenopus

Dev. Biol.

Regular Article
Properties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression