Abstract
The time between embryonic days 11 and 15 (E11--E15) in the developing mouse spinal cord is a critical period of neuronal patterning during which cellular proliferation slows as populations of neuronal precursors differentiate, migrate, and form primitive electrical circuits underlying the future sensory and neuromuscular systems. Some of the key molecular factors that direct these events have been identified including SHH, BMPs, and members of the bHLH and homeodomain gene families. Despite significant progress, many unidentified molecular factors likely exist, operating in pathways that remain poorly understood or completely unknown. Identification of these factors and their placement into molecular networks is a challenge requiring individual gene studies as well as genomic approaches. In a genomic approach using DNA microarrays, this investigation profiled transcriptional changes during the E11--E15 window as a function of time and rostral-caudal location in the embryonic mouse spinal cord. We identified 818 differentially expressed genes in comparisons of E15 and E11 whole spinal cord. Ontological segmentation of this group reveals striking differences between the down and up-regulated genes with down-regulated genes (E15 relative to E11) largely involved in cellular proliferation processes (e.g., mitosis, DNA replication and the cell cycle); while the up-regulated list is heterogeneous with over-representation of transport, signaling and neural-specific gene groups. To refine our characterization of gene expression we performed additional microarray experiments in a factorial design in which E13 and E15 cervical and lumbar regions were compared to like regions from E11 spinal cords, and time-matched cervical and lumbar regions were directly compared at E11, E13, and E15. Quantification of mRNA differences with real time PCR on 11 genes across 3 experimental conditions was found to be in excellent agreement with the microarray results, validating the data set. In-situ hybridizations of four genes (DNER, GPM6b, Syngr3 and Syt4) demonstrated dynamic patterns of expression during this time period, suggesting that these genes are functionally significant in the development of the spinal cord. The aggregate data were incorporated into a linear model that predicts gene expression as a function of time and rostral-caudal location in the embryonic spinal cord during the E11--E15 window. Clustering of these data by expression profile reveals distinct groups including a Hox gene ensemble that corroborates previous reports and suggests novel spatiotemporal expression patterns. This model serves to expand our knowledge of the molecular details of spinal cord development during a central window of neural patterning and provides insight into related processes such as the directed differentiation of neural stem cells.