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. 2012 Aug 1:13:90.
doi: 10.1186/1471-2202-13-90.

Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development

David A Cameron  1 , Frank A Middleton, Anjen Chenn, Eric C Olson
Affiliations

Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development

David A Cameron et al. BMC Neurosci. .

Abstract

Background: Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled.

Results: We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate.

Conclusions: This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.

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Figures

Figure 1
Figure 1
Isolation, profiling, and QISP analysis of GFP + (Eomes+) cortical neurons. a, Neocortex (NCX) containing GFP + neurons is dissected, and cells isolated with flourescence activated cell sorting (FACS). Following transcriptional profiling, genes are sorted by RMA and their level of fold up- or down-regulation compared to the FACS GFP- population. Verification of up-regulated genes is accomplished with quantified in situ hybridization (QISP), and the verified genes hierarchically clustered (see Methods). b, Fluorescence microscopic images of a section from an E14.5 Eomes::eGFP brain, displaying GFP + (green) and GFP- cells, that are blue due to Hoechst nuclear labeling. Subregions of Interest (sROIs) 1-20 are superimposed in white on the image. Scale bar, 25 μm. MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone; LGE, lateral ganglionic eminence. c, Representative outcome of FAC sort of GFP + and GFP- cells. A GFP + (P3) and GFP- (P4) were isolated based on fluorescence (GFP-A) and side scatter (SSC-A). The boxes denote the relevant sorted populations. All in situ hybridizations were obtained from Genepaint.
Figure 2
Figure 2
Comparison of RMA values between samples. a, a’, Comparison of RMA values between E14.5 GFP + and E13.5 GFP + cells. b, b’) Comparison of RMA values between E14.5 GFP + and E13.5 GFP- cells.
Figure 3
Figure 3
Identification and hierarchical clustering of QISPs. a, Hierarchical clustering of 317 QISPs representing transcripts expressed ≥ 3 fold higher in Eomes + (GFP+) neurons, compared to GFP- cells. From left to right: gene clusters denoted numerically (1-15); expression levels in the MZ + CP, IZ, and VZ (red indicates high expression, green indicates low expression) and gene number (1-317). b, Gene lists of the clustered QISPs from panel a. QISPs were imported into Multiple Experiment Viewer (MeV) and clustered using Pearson’s correlation (see Methods). White and shaded areas demarcate the different gene clusters.
Figure 4
Figure 4
Transcripts encoding transcription factors. Functional groupings of QISPs based upon Gene Ontology (GO) molecular function and biological process annotations. a, Clustered QISPs of transcripts expressed ≥ 3 fold higher in Eomes + neurons compared to GFP- cells. The left-most dendrogram reveals the hierarchical arrangement of QISPs. b, Clustered QISPs of transcripts expressed ≥ 3 fold lower in Eomes + neurons compared to GFP- cells. a’ and b’.
Figure 5
Figure 5
Network analyses of dynamically regulated transcription factors (TFs) in differentiating cortical excitatory neurons, derived from the IPA-Ingenuity Network Tool (see Methods). a, ≥ 3 fold up- and ≥ 3 fold down-regulated TFs were imported along with endogenous chemicals (e.g., retinoic acid) to populate the network. Grey boxes collect the up-regulated genes expressed in the “upper,” “mid” and “deep” cortical regions (CP + MZ, IZ, VZ alone) as well as the down-regulated genes (VZ) and the group of IPA Interacting Genes. Gray lines that are numbered indicate interactions between cortical zones (upper, middle, and deep); light blue lines indicate direct interactions of upper, middle, and deep up-regulated genes, and down-regulated genes, with IPA interacting genes. Dark blue lines indicate interactions between IPA interacting genes (see Results). b, Tabular summary of interactions between cortical zones. 1, Interaction identified by IPA Ingenuity analysis.
Figure 6
Figure 6
Transcripts encoding receptors. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes:: GFP + cells compared to eGFP- cells.
Figure 7
Figure 7
Transcripts encoding synaptic proteins. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher in Eomes-GFP + cells compared to GFP- cells. Note: there were no downregulated synaptic transcripts.
Figure 8
Figure 8
Transcripts encoding ion channels and solute carriers. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes-GFP + cells compared to GFP- cells.
Figure 9
Figure 9
Transcripts encoding adhesion molecules. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes-GFP + cells compared to GFP- cells.
Figure 10
Figure 10
Schematic of excitatory neuron migration, differentiation, and patterns of gene expression. a, Illustration of radial migration, and general morphological characteristics, of excitatory neurons. RGC, radial glial cell; IPC, intermediate precursor cell; MPN, multipolar neuron; BPN, bipolar neuron; PMN, postmigratory neuron; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. b, Pie charts representing gene clusters 1-14 (see Figure 3) as a function of their relative contribution to the population of ≥ 3 fold up-regulated genes as a function of cortical location (upper, middle, deep). The accompanying table (bottom) indicates the Gene Ontology (GO) classification for each of the gene clusters, and the percentage of genes within each GO classification in each zone (D, M, U). c, The individual, greatest up-regulated genes in the Eomes lineage for each cortical zone.
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