1Identification of genes differentially expressed in the embryonic pig cerebral cortex before and after appearance of gyration

Abstract

Background: Mammalian evolution is characterized by a progressive expansion of the surface area of the cerebral cortex, an increase that is accompanied by gyration of the cortical surface. The mechanisms controlling this gyration process are not well characterized but mutational analyses indicate that genes involved in neuronal migration play an important function. Due to the lack of gyration of the rodent brain it is important to establish alternative models to examine brain development during the gyration process. The pig brain is gyrated and accordingly is a candidate alternative model.

Findings: In this study we have identified genes differentially expressed in the pig cerebral cortex before and after appearance of gyration. Pig cortical tissue from two time points in development representing a non-folded, lissencephalic, brain (embryonic day 60) and primary-folded, gyrencephalic, brain (embryonic day 80) were examined by whole genome expression microarray studies. 91 differentially expressed transcripts (fold change >3) were identified. 84 transcripts were annotated and encoding proteins involved in for example neuronal migration, calcium binding, and cytoskeletal structuring. Quantitative real-time PCR was used to confirm the regulation of a subset of the identified genes.

Conclusion: This study provides identification of genes which are differentially expressed in the pig cerebral cortex before and after appearance of brain gyration. The identified genes include novel candidate genes which could have functional importance for brain development.

Figure 1

Expression analysis of genes involved in mammalian brain convolution. mRNA was extracted from pig cortical tissue from E60 and E80. qRT-PCR analysis were performed on cDNA for the genes DCX, ARX, GPR56, FLNA, Reelin, VLDLR, ApoER2, Dab1, FYN, LIS1, NDEL1, and CDK5. The expression levels were normalized to GAPDH, Beta-actin and 18S rRNA expression using the geNorm program [30].

Figure 2

Verification of microarray data with qRT-PCR. Nine genes differently expressed from the microarray analysis were examined for the expression level using mRNA extracted from pig cortical tissue from E60 and E80. qRT-PCR analysis were performed on cDNA for the genes GFAP, ApoE, calbindin-2, Neurofilament (200 kDa), S100A1, TUBA1, Neurogranin, ACTN2, CHN1, and Dbx17. The expression levels were normalized to GAPDH, Beta-actin and 18S rRNA expression using the geNorm program [30].