Identification of a neuronal gene expression signature: role of cell cycle arrest in murine neuronal differentiation in vitro

Abstract

Stem cells are a potential key strategy for treating neurodegenerative diseases in which the generation of new neurons is critical. A better understanding of the characteristics and molecular properties of neural stem cells (NSCs) and differentiated neurons can help with assessing neuronal maturity and, possibly, in devising better therapeutic strategies. We have performed an in-depth gene expression profiling study of murine NSCs and primary neurons derived from embryonic mouse brains. Microarray analysis revealed a neuron-specific gene expression signature that distinguishes primary neurons from NSCs, with elevated levels of transcripts involved in neuronal functions, such as neurite development and axon guidance in primary neurons and decreased levels of multiple cytokine transcripts. Among the differentially expressed genes, we found a statistically significant enrichment of genes in the ephrin, neurotrophin, CDK5, and actin pathways, which control multiple neuronal-specific functions. We then artificially blocked the cell cycle of NSCs with mitomycin C (MMC) and examined cellular morphology and gene expression signatures. Although these MMC-treated NSCs displayed a neuronal morphology and expressed some neuronal differentiation marker genes, their gene expression patterns were very different from primary neurons. We conclude that 1) fully differentiated mouse primary neurons display a specific neuronal gene expression signature; 2) cell cycle block at the S phase in NSCs with MMC does not induce the formation of fully differentiated neurons; 3) cytokines change their expression pattern during differentiation of NSCs into neurons; and 4) signaling pathways of ephrin, neurotrophin, CDK5, and actin, related to major neuronal features, are dynamically enriched in genes showing changes in expression level.

Fig. 1.

Characteristics of neural stem cells (NSCs) and neurons. A: NSCs were cultured in 21% O₂-5% CO₂. Cells were costained by using antibodies directed against mouse stem cell marker nestin and neuronal markers MAP2 (a–d) or against nestin and the neuronal marker NeuN (e–h). All cells expressed nestin (b, f), but there was no detectable MAP2 or NeuN (a, e). Phase contrast (Ph. Contr.) microscopy revealed a pyramidal shape of the cells (i), and only very few dead cells were observed or were seen with propidium iodide (PI) labeling (j). Magnification: ×100; insets, ×400. Western blot analysis confirmed the absence of MAP2 and NeuN from NSCs (k). B: primary neurons were cultured from embryonic day 17 mouse cortex in normoxia for 8 days. Immunocytochemistry was performed for nestin, MAP2, and NeuN. There was a complete absence of nestin (b). MAP2 (a, e), and NeuN (f) were detected in all cells. Phase contrast microscopy revealed typical neuronal shape with processes and synapses (i). Magnification: ×100; insets, ×400. Western blot analysis confirmed the presence of MAP2 and NeuN and the absence of nestin from primary neurons.

Fig. 2.

Microarray analysis of NSCs and neurons. A: number of microarray probes up- and downregulated comparing NSC ± MMC and primary isolated neurons. MMC, mitomycin C. B: heat plot representing all possible pairwise hierarchical ordered partitioning and collapsing hybrid (HOPACH) correlation (euclidean) values. Darkest values indicate a high level of correlation (= 1 across the diagonal), and lighter gray indicates lower correlation values.

Fig. 3.

Functional annotation of transcripts differentially expressed between NSC ± MMC and primary neurons. HOPACH cluster of the 5,488 microarray probes that where differentially expressed (F-test statistic P < 0.05 and a 2-fold change in up- or downregulated compared with NSC control in any of the 2 comparisons). Clusters where annotated for statistical overrepresentation of transcript-associated Gene Ontology (GO) terms using MAPPFinder and GO-Elite (14) software.

Fig. 4.

Biofunctional analysis of neuron gene expression profile relative to NSCs. Functional characterization was done using the Ingenuity Pathway Analysis software. Genes significantly altered were classified into associated functions (as depicted in the x-axis). Functions are listed from most to least significant (left to right). The y-axis depicts −log₁₀ [P value]. Horizontal bar (threshold) indicates P < 0.05. The significance threshold was set to 1.3. Primary neurons: a total of 53 biofunctions were significantly changed (shown are the first 17 most significant pathways). MMC-treated NSCs; a total of 17 biofunctions were significantly changed.

Fig. 5.

Ephrin and neurotrophin signaling pathways in primary neurons. Analysis using the Ingenuity Pathway Analysis software showed that ephrin (A) and neurotrophin (B) are among the pathways that control the highest number of neuronal-specific functions and showed a majority of genes have change in gene expression. Red, upregulated; green, downregulated; white, no change. Ephrin pathway: dynamic change in gene expression (up- and downregulation) is observed for molecules related to growth cone collapse, axon retraction, and cell repulsion, with upregulation of genes related to cell growth, dendritic spine morphogenesis, synaptic plasticity, and axon guidance. Neurotrophin pathway: there is a downregulation of genes related to apoptosis, and upregulation of genes related to synaptic plasticity; predominantly upregulation of genes related to neurite outgrowth.

Fig. 6.

CDK5 (A) and actin (B) signaling pathway in primary neurons. Analysis using the Ingenuity Pathway Analysis software showed that CDK5 and actin signaling pathways are involved in multiple functions related to neurons, and a majority of the genes in those pathways have their expression level changed. Red, upregulated; green, downregulated; white, no change. CDK5: pathway shows upregulation of multiple genes related to neurite outgrowth, neurotransmitter receptors and ion channels. Actin: there is a dynamic change in the expression of genes related to actin polymerization and focal adhesion, suggesting dynamic instability of actin cytoskeleton, a feature known to be needed for neurotransmitter secretion, and axon guidance.

Fig. 7.

Generation and characterization of neuronal cells after MMC induction of NSCs. C17.2 NSCs were treated with 0.4 μg/ml of MMC under normoxic conditions, and immunostaining for the stem cell marker nestin and early neuronal marker NeuN is shown at 2 time points: 1 day and 6 days after treatment (A). Nestin was detected at day 1 (A, b) and decreased in intensity afterwards but remained detectable (A, f). MAP2 was weakly expressed at day 1 (A, a) and increased in intensity afterwards (A, e). Western blot analysis showed 2-fold increase in MAP2 from day 1 to day 3 and a significant (P < 0.005) ∼5-fold increase of NeuN from day 1 to day 6 (A, i, j). After 6 days of culture when the phenotype stabilized, cells were further cultured for an additional 2 days (total 8 days) and then characterized similarly. They were stained for nestin (B, b), MAP2 (B, a, e), and NeuN (B, f). Phase contrast microscopy confirmed the neuronal morphology (B, i). Mortality was only at a baseline level (B, j). Magnification: ×100; insets, ×400. Student's t-test, NeuN level at day 1 vs. day 6, P < 0.005.

Fig. 8.

Maintenance of neuronal phenotype in a primary neuron: suggested model. Molecules at the cell surface represent receptors and/or ligands, as indicated. Molecules underlined are upregulated, and molecules in italics are downregulated, in microarray analysis. Some major neuronal-specific functions and the upstream signals are involved in its maintenance. Growth factors are indicated in boldface type, since some are up- and some are downregulated (see Table 3). All other molecules are not changed on the microarray analysis. GF, growth factors; AF, axon formation; AG, axon guidance; AR, axon remodeling; AA, anti-apotptosis; PR, proliferation response; axon guid, axon guidance; LTP, long-term potentiation; SFA, stress fiber activation; GluR. glutamate receptor; AGC, attraction of growth cone.

Fig. 9.

Maintenance of cell cycle block in primary neurons. Microarray analysis revealed changes in genes expression of molecules involved in cell cycle that were either downregulated (italics) or upregulated (underlined). Downregulated molecules include factors involved in promoting cell cycle, such as cyclins and CDKs, and upregulated molecules were mostly antiproliferative, such as Rb and p21. The pattern of change in the level of these molecules can maintain a block in cell cycle at two levels: G₁/S block and G₂/M block, which will keep the cell nondividing, despite the presence of stimulatory growth factors.

Fig. 10.

Major pathways involved in survival and growth in primary neurons. Microarray analysis revealed changes in gene expression levels of many cytokines (that were mainly downregulated, in italics), and growth factors that were mainly upregulated (underlined). The data showed molecules that can induce apoptosis and cell division were downregulated, and those that can induce cell cycle block were upregulated. Many growth-promoting factors were upregulated, leading to downstream pathways that favor protein synthesis, cellular growth, and cytoskeleton rearrangements. IKβ, inhibitor of κB; IKKβ, inhibitor of κB kinase.

Fig. 11.

Gene expression changes maintain the phenotype of a differentiated neuron. A proposed model for how changes in gene expression that take place when a NSC differentiates into a neuron and maintains the phenotype of the mature neuron. Changes in cell cycle genes maintain a permanent block of cell division. Changes in cytokines (Cyto.) and growth factors (GF) assure the survival and growth of the cell; Finally, neuronal-related pathways are activated, and changes in gene expression of many genes in those pathways assure the presence of the basic constituents of a neuron, such as neurotransmitter, neurite, polarity, etc.