Autism-Associated Gene Engrailed-2 Plays a Cell Autonomous Role in Regulating Proliferation and Cell Death in Hippocampal Neural Stem Cells in Vitro
Objectives: This study seeks to examine cell autonomous roles for En2 in regulating hippocampal neurogenesis by showing that (1) stem cells cultured in vitro as neurospheres express En2, and (2) the absence of En2in cultured cells leads to increased proliferation and apoptosis.
Methods: Hippocampi from P7 En2-KO and WT mice were dissociated and cells were plated in defined media containing 2% B27 supplement, 20 ng/ml EGF and 10 ng/ml FGF. En2 mRNA expression was assessed using quantitative PCR. Primary neurospheres were assayed for neurosphere numbers and size after 7 days in culture. Immunofluorescent labeling of whole neurospheres was performed to determine rates of proliferation (BrdU incorporation) and apoptosis (cleaved caspase-3, pyknosis). Experiments were performed at least 3 times, with each experiment consisting of at least one WT and En2-KO culture.
Results: Expression of En2 mRNA as well as its homolog Engrailed-1 was detected in WT neurospheres. While primary neurosphere assays revealed no change in numbers of primary spheres, measurement of sphere diameter showed increased frequency of larger spheres in En2-KO, suggesting potential effects on proliferation and/or survival. Compared to WT neurospheres, the En2-KO exhibited a 2-fold increase in BrdU labeling (p<0.005) as well as apoptosis (p<0.05).
Conclusions: This study suggests that En2 plays a role in regulating proliferation and apoptosis in hippocampal neural stem cells. While evidence of En2 expression in hippocampal dentate gyrus cells in vivo remains uncertain, we were able to show expression of En2 in neural precursor cultures. Furthermore, increased proliferation and cell death observed in En2-KO neurosphere cultures recapitulates what is observed in vivo, suggesting that En2 may inhibit proliferation and promote survival in a cell autonomous manner. More broadly, these studies indicate how the activities of developmental regulatory genes in diverse brain regions impact brain structure and function, and may contribute to our understanding of how ASD-related behaviors occur in the En2-KO mouse.