Transcriptomic Analysis of CAD Cell Differentiation

Abstract

CAD cells were derived from Cath.a cells, a mouse central nervous system catecholaminergic cell line. Serum-starved CAD cells undergo morphological changes and resemble isolated neurons when observed by microscopy. We carried out an RNAseq transcriptomic analysis to examine differentiated CAD cells for expression signatures related to neuronal functions, identifying ~1900 transcripts whose expression changed with differentiation. Pathview analysis identified ~80 KEGG pathway gene sets that were differentially expressed, including upregulation of at least 13 neuron-related pathways. This dataset can be explored more deeply, allowing further investigation into expression changes relevant to studying neuronal functions in this easy-to-culture model system.

Figure 1:. CAD cells differentiate into neuron-like cells with serum deprivation.

The cell morphology of CAD cells changes to become more neuron-like after 5 days of growth in serum-free media. Undifferentiated cells form lamellae and sit flat or in clumps, while differentiated cells have long projections that form networks. (A) Phalloidin staining for actin is shown in yellow and DAPI staining for DNA is shown in magenta as visualized by fluorescence microscopy. (B) Representative scanning electron microscopy images. Displayed images are representative of commonly observed phenotypes. All scale bars are 20 µm. (C) Principal component analysis (PCA) indicated that 89% of the variation between RNAseq samples of differentiated and undifferentiated CAD cells could be attributed to differentiation state of the cells. (D) After 5 days of differentiation, 1445 genes were upregulated and 451 genes were downregulated with a more than 2-fold change in gene expression. Red markers outside of the shaded area indicate expression-level changes greater than 2-fold in magnitude with a p_adj ≤ 0.1. (E) Pathview analysis revealed differential expression for a subset of KEGG pathway gene sets compared to undifferentiated CAD cells, including upregulation of 13 gene pathways related to neuronal function (q ≤ 0.1).