Temporal Gene Expression Profiling of Astrocyte Precursors During Development

Lineage development is a step-wise process, governed by stage-specific regulatory factors and associated markers. Astrocytes are one of the principle cell types in the CNS and the stages associated with their development remain very poorly defined. To identify these stages we performed gene expression profiling on astrocyte precursor populations in the spinal cord, identifying distinct patterns of gene induction during their development. Validation studies identified a new cohort of astrocyte-associated genes during development and demonstrated their expression in reactive astrocytes in human white matter injury (WMI). To begin dissecting the intermediate stages of astrocyte development, we performed in vivo temporal gene expression profiling of astrocyte precursor cells from the spinal cord of Glast-dsRed mice. Glast is a glutamate transporter and its induction coincides with the gliogenic switch and continues into mature astrocytes in the spinal cord. We chose 6 time points between mE12.5, the initiation of gliogenesis, and mP7, an early post-natal stage where astrocytes express mature markers. During this developmental interval, Glast-expressing cells emerge from the VZ, populating both the mantle zone and the white matter. However, a subset of Glast-expressing cells remains in the ventricular zone (VZ), where progenitor cells are located, marking cell populations in various lineages and differentiative states during spinal cord development. To sub-fractionate prospective Glast-expressing astrocyte precursors from other potential Glast-expressing lineages, we used the cell surface marker LexA/CD15, which has been used previously to purify astrocytes and various progenitor populations. Upon dissection of glast-dsRed spinal cords, we incubated dissociated cells with CD15 antibody prior to FACS isolation. We isolated 3 populations at each time point: Glast-dsRed+/CD15- (henceforth +/-), Glast-dsRed+/CD15+ (henceforth +/+), Glast-dsRed-/CD15- (henceforth -/-). Please note that "-/+" and "+/-" are the same.