Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

doi:10.1016/S0304-3940(96)13214-6

Neuroscience Letters

Volume 220, Issue 2, 13 December 1996, Pages 81-84

https://doi.org/10.1016/S0304-3940(96)13214-6 Get rights and content

Abstract

Cerebellar granule cells isolated from postnatal day 7 mice, and cultured in minimal medium containing only insulin-like growth factor-I (IGF-I), both survive and differentiate. This differentiation is marked by neurite growth and expression of genes associated with terminal differentiation, the myocyte-specific enhancer factor 2A (MEF2A) and the α6 subunit of the γ-aminobutyric acid_A receptor (GABA_Aα6). Percoll gradient purified granule cells maintained without IGF-I, in minimal medium alone or in medium containing the antioxidant N-acetylcysteine (NAC), also express MEF2A and GABA_Aα6. Thus, cultured granule neurons can differentiate to some extent cell-autonomously and IGF-I may not be a critical factor for this process.

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Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes, but how TFs dynamically respond to paralog depletion on a genome-wide scale in vivo remains incompletely understood. Using single and double conditional knockout of myocyte enhancer factor 2 (MEF2) family TFs in granule neurons of the mouse cerebellum, we find that MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. Although both TFs are highly expressed in granule neurons, transcriptomic analyses show MEF2D is the predominant genomic regulator of gene expression in vivo. Strikingly, genome-wide occupancy analyses reveal upon depletion of MEF2D, MEF2A occupancy robustly increases at a subset of sites normally bound to MEF2D. Importantly, sites experiencing compensatory MEF2A occupancy are concentrated within open chromatin and undergo functional compensation for genomic activation and gene expression. Finally, motor activity induces a switch from non-compensatory to compensatory MEF2-dependent gene regulation. These studies uncover genome-wide functional interdependency between paralogous TFs in the brain.
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Thus, injection of a HA (Fig. 5; circle B) may trigger BH3-only sensitizer activation that inhibits Bcl-xL enough to allow loaded activated Bax to initiate apoptosis. Alternatively, when EGL NPCs differentiate (rather than undergo apoptosis) in vitro, they are cultured in Hh-free medium (Lin and Bulleit, 1996; Wechsler-Reya and Scott, 1999). As a result, there is no initial strong Hh stimulation to load activated Bax (priming them for death) before the abrupt loss of Hh signaling.
The external granule layer (EGL) is a proliferative region that produces over 90% of the neurons in the cerebellum but can also malignantly transform into a cerebellar tumor called the medulloblastoma (the most common malignant brain tumor in children). Current dogma considers Hedgehog stimulation a potent proliferative signal for EGL neural progenitor cells (NPCs) and medulloblastomas. However, the Hedgehog pathway also acts as a survival signal in the neural tube where it regulates dorsoventral patterning by controlling NPC apoptosis. Here we show that Hedgehog stimulation is also a potent survival signal in the EGL and medulloblastomas that produces a massive apoptotic response within hours of signal loss in mice. This toxicity can be produced by numerous Hedgehog antagonists (vismodegib, cyclopamine, and jervine) and is Bax/Bak dependent but p53 independent. Finally, since glucocorticoids can also induce EGL and medulloblastoma apoptosis, we show that Hedgehog's effects on apoptosis can occur independent of glucocorticoid stimulation. This effect may play a major role in cerebellar development by directing where EGL proliferation occurs thereby morphologically sculpting growth. It may also be a previously unknown major therapeutic effect of Hedgehog antagonists during medulloblastoma therapy. Results are discussed in terms of their implications for both cerebellar development and medulloblastoma treatment.
Self-inactivating lentiviruses: Versatile vectors for quantitative transduction of cerebellar granule neurons and their progenitors
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Cerebellar granule neurons (CGNs) undergo a well-defined, intrinsic differentiation program that is recapitulated in vitro. Thus, homogeneous cultures of CGNs provide an excellent opportunity to define the mechanisms underlying their development. The ability to alter endogenous gene expression in CGNs on a population-wide basis would greatly facilitate the elucidation of these events. In the present study, we show that self-inactivating lentiviruses efficiently infect both dividing progenitors and post-mitotic CGN cultures in a quantitative manner without altering their cellular properties. The time course for protein expression was biphasic for both types of cultures, with the first peak occurring during the initial infection period. Thus, lentiviruses can express proteins in CGNs both acutely and on a long-term basis to study developmental and other processes continuously over an extended time period. These vectors also infected CGNs in cerebellar slice preparations. In addition, lentiviruses harboring a transgene for the mouse GABA_A receptor α6 subunit promoter recapitulated the differentiation-dependent expression of this gene in CGN cultures. Self-inactivating lentiviruses are extremely versatile vectors that offer important advantages for studies of protein function and gene regulation. The ability to alter protein function on a global scale in CGN cultures permits biochemical assessment of its impact on mRNA and protein populations, as well as on protein–protein and protein–DNA interactions. Further, integrated lentiviruses can be used to study chromatin-dependent promoter regulation and transcription factor interactions in CGNs over time in a facile manner.
A role for nuclear factor i in the intrinsic control of cerebellar granule neuron gene expression
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Similarly, granule neurons of HNF-3β transgenic mice migrate inappropriately and do not form a normal internal granule cell layer, yet continue to express GABRA6 (38). Numerous studies also have shown that the expression of GABRA6 is retained in isolated CGN cultures (6, 29). The present studies demonstrate that NFI is an integral part of the intrinsic program directing CGN differentiation through its regulation of the GABRA6 gene.
Nervous system formation requires the elaboration of a complex series of differentiation events in both a spatially and maturation-regulated manner. A fundamental question is how neuronal subtype specification and developmental gene expression are controlled within maturing neurons. The α6 subunit of the γ-aminobutyric acid type A (GABA_A) receptor (GABRA6) is preferentially expressed in cerebellar granule neurons and is part of an intrinsic program directing their differentiation. We have employed a lentiviral approach to examine the transcriptional mechanisms controlling neuronal subtype-selective expression of this gene. These studies demonstrated that nuclear factor I (NFI) proteins are required for both transgenic GABRA6 promoter activity as well as endogenous expression of this gene in cerebellar granule neurons. Chromatin immunoprecipitation also showed that NFI proteins are bound to the GABRA6 promoter in these cells in vivo. Furthermore, analyses of gene knockout mice revealed that Nfia is specifically required for normal expression of the GABRA6 gene in cerebellar granule neurons. NFI expression and DNA binding activity are highly enriched in granule neurons, implicating this transcription factor family in the neuronal subtype-selective expression of the GABRA6 gene. These studies define a new role for NFI proteins as neuronal subtype-enriched transcriptional regulators that participate in an intrinsic transcriptional program directing the differentiation of cerebellar granule neurons.
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Citation Excerpt :
A minimal culture environment allows cerebellar granule neurons to undergo terminal differentiation [28–30]. We previously observed that purified granule cells, cultured with insulin-like growth factor I (IGF-I) as the only added trophic factor, survive and after 3–5 days of culture express mRNA encoding the GABAA receptor α6 subunit [28, 29]. BDNF added to these cultures, in the presence or absence of IGF-I, accelerated granule neuron maturation with GABAAα6 expression occurring 1–2 days after the start of culture [30].
Brain-derived neurotrophic factor (BDNF) can regulate the maturation of developing cerebellar granule neurons. Within 1–2 days of culture, BDNF induces the expression of granule neuron terminal differentiation markers, particularly GABA_A receptor α6 subunit (GABA_Aα6) mRNA. Other trophic factors including insulin-like growth factor, the neurotrophin NT-3, pituitary adenylate cyclase-activating polypeptide (PACAP), and fetal bovine serum failed to induce this early expression. The expression of other GABA_A receptor subunits, including α1 and γ2, was also enhanced by exposure of developing granule neurons to BDNF. This BDNF-dependent expression of GABA_A receptor subunit mRNAs could be effectively blocked by treatment with the mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 or U0126. In the absence of BDNF, GABA_Aα6 expression occurs but not until 3–4 days of culture. This BDNF-independent expression of GABA_Aα6 was also inhibited by PD98059. Further studies showed that the BDNF-dependent expression GABA_Aα6 could also be reduced by LY294002, an inhibitor of the phosphatidylinositol 3-kinase, or depolarizing concentrations of KCl. These results thus suggest that both BDNF-dependent and -independent expressions of GABA_A receptor subunits require the activation of MEK and the mitogen-activated protein kinase (MAPK) pathway. However, it is also likely that other signaling pathways modulate this maturation process.
Control of neuronal precursor proliferation in the cerebellum by sonic hedgehog
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Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

Abstract

Neuron

Free Radical Biol. Med.

Cell

Neuron.

J. Mol. Biol.

Exp. Eye Res.

J. Biol. Chem.

Transient IGF-I gene expression during the maturation of functionally related central projection neurons

J. Neurosci.

NMDA receptor activation in differentiation cerebellar cell culture regulates the expression of a new POU gene, Cns-1

J. Neurosci.

Neurotrophin-45 (NT-45) and brain-derived neurotrophic factor (BDNF) act at later stages of cerebellar granule cell differentiation

J. Neurosci.

Neuronal regulation of astroglial morphology and proliferation in vitro

J. Cell Biol.

$Neurotrophin - 45 (NT - 45)$ and brain-derived neurotrophic factor (BDNF) act at later stages of cerebellar granule cell differentiation