TGF-beta, COUP-TFII, and Gli1/Zic transcription factor dynamics revealed in Retinoic acid induced Neural Tube Defect

The morphologic changes of neuroepithelial cells, and their interactions with surrounding cells, are fundamental to primary neurulation and are a function of several timed signaling gradients. Retinoic acid administration at fetal day E10 induces neural tube defects in 84.2% of rat pups. Negative selection using A2B5 and E-NCAM has been validated for isolation of neuroepithelial cells for culture. Here we report the isolation and analysis of the fundamental actors in primary neurulation by flow cytometry with CD147 positive selection followed by whole transcriptome analysis of this purified population via microarray enhanced with the ERCC RNA internal controls. Comparison of the gene expression in Retinoic acid exposed versus wild-type isolates shows excellent correspondence to known neural tube defect genes. Analysis of transcription factor binding sites in regulatory regions of differentially expressed genes, implicates a binding site “cross-roads” where improper signaling through a multitude of pathways could potentially elicit neural tube defects. Six samples total, three replicates each, of pooled biological replicates. ERCC ExFold mix 1 added to CTR ERCC ExFold mix 2 added to RA.