Abstract
Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.
Highlights
Astrocytes are among the most abundant cells in the brain [1] and contribute to a multitude of complex and essential functions
neural progenitor cell (NPC) were derived from human embryonic stem cells (hESCs) through sequential formation of embryonic bodies, rosettes, and neurospheres (Fig. 1A, panels a–e), which differentiated into star-shaped astrocytes (Fig. 1A, panels f–h)
Astrocyte differentiation was confirmed by immunostaining for glial fibrillary acidic protein (GFAP) (Fig. 1A, panels i and j), an intermediate filament protein that helps to maintain the mechanical strength of astrocytes [23] and is highly expressed on reactive astrocytes [24, 25], as well as by co-immunostaining for GFAP and S100 calciumbinding protein b (S100b) (Fig. S1), a neurotrophic protein that acts as an autocrine factor promoting astrocyte proliferation [26, 27]
Summary
Astrocytes are among the most abundant cells in the brain [1] and contribute to a multitude of complex and essential functions. We demonstrate that PITX1 expression increased during human astrocyte differentiation and that activation of the SOX9 promoter induced by PITX1 is critical for this process. We examined CD44 and GFAP protein expression levels by Western blotting to confirm astrocyte differentiation. Western blotting indicated that GFAP expression was eliminated in PITX1knockdown cells but was up-regulated in cells transfected with scrambled shRNA during astrocyte differentiation (Fig. 3, B and C).
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