Figure 1.
Light pathway within the SCN and genes that involved in the process.
A) An illustration of light signal's progression through the core and shell of the SCN. Major functional groups are highlighted including membrane receptors, cellular signal pathways, immediate early gene activations, and circadian oscillator. B) A consolidated view of an SCN neuron. Genes listed were used in the qPCR assay, and focus on the circadian clock and genes involved in mediating its response to light. Definitive gene functions are shown in solid arrows. Implied gene functions are shown in dashed arrows.
Figure 2.
Laser Capture Microdissection in Mouse SCN.
A cryostat section through the mouse SCN was stained with hematoxylin and counterstained with eosin. Top, dry-mounted brain section before LCM capture. The SCN is identified by the location of the optic chiasm (OC) and the third ventricle (3v) as well as higher neuronal density apparent by staining. Middle, the core region (arrows, ventral-lateral part of the SCN) has been captured. Bottom, the shell region (arrowheads; dorsal-medial part of the SCN has been captured.
Figure 3.
Hierarchical clustering of laser captured mouse SCN samples and principal component analysis.
Results from qPCR were normalized by calculating −ΔCT using the average CT of gapdh, tbp, and actb as control. A) Unsupervised clustering of samples based on similar expression profiles showing separation between brain regions, times, and light treatment. Gray bars represent failed PCR reactions. B) PCA of the SCN samples in PC1 and PC2 space. Colors and shapes represent the same as in A). Clear separations between the SCN core and shell samples as well as the light pulse core samples are circled. C) Calculated eigenvector values of the first two 2 PCs. Each dot represents a gene. Genes with greater influence on PCA (loading factor greater than 0.15 or less than −0.15) are shown in pink with the names listed. D) Expression of these genes with more extreme loading factors are also shown in hierarchical cluster based on Pearson Correlation. Each row represents a gene and each column represents one LCM sample. Samples are grouped by regions (core and shell of the SCN, hypothalamus), then by time of collection (ZT 6, ZT 15, and ZT 15 with light pulse (ZT 15+LP)). Color bar represent a median centered −ΔCT range of −3 to 3.
Figure 4.
Distinct temporal and spatial expression profiles in core and shell of the SCN and effects of light pulse.
A) PCA of non-light-pulsed SCN core and shell samples is shown in PC1 and PC2 space. Clear separations of core and shell samples by PC1 values as well as day (ZT 6) and night (ZT 15) samples by PC2 values can be seen. Light pulsed samples (red, ZT 15+LP) are projected based on calculated eigenvectors of the first 2 PCs. Solid arrow indicates trend of light-pulsed shell samples. Dashed arrow indicates trend of light-pulsed core samples. B) Calculated eigenvector values of first two 2 PCs. Each dot represents a gene. Genes with greater influence on PCA (loading factor greater than 0.15 or less than −0.15) are shown in pink with the names listed.
Figure 5.
Differential Expression in SCN.
Expression differences (−ΔΔCT) are shown. A) The presence or absence of a light pulse at ZT 15 significantly affected the expression of 32 genes in the SCN. The genes are ordered based on the −ΔΔCT values. Genes significantly expressed in either sub-panel are shown in both sub-panels. B) A total of 29 genes showed significant differential expression between core and shell of the SCN. C) A total of 26 genes showed significant differences between day (ZT 6) and night (ZT 15). Black columns indicate significant differences (ANOVA with post hoc t-test, p<0.05). Error bars indicate standard errors.
Figure 6.
Functional networks of enriched gene sets in the SCN.
The genes selected for qPCR were annotated using DAVID. Relevant functional terms were manually selected. A) Network representation of term interactions was generated using Cytoscape. The node size represents the number of genes that have been annotated to a particular term. The connection between the nodes means at least one gene is shared between the two terms with greater number of shared genes represented by thicker connections as the scale bars indicated. B) A network based on shell-enriched genes. C) A network represents the genes elevated during the day (ZT 6). D) A network represents the genes elevated during the night (ZT 15). E) A network based on light-induced gene only.
Table 1.
TRE enrichment analysis of light-induced genes.
Figure 7.
Co-regulation of light-induced gene activation.
TRE families (diamonds) that are significantly enriched and genes (rounded rectangles) they regulate. Genes with promoters that shared the same TRE pattern are consolidated. Confirmed light induced genes are shown in red. Arrow lines indicate the presence of TRE in the promoter regions of a group of genes.