In a previous study screening libraries we could demon strate that inhibition of class I and II HDACs with TSA leads to an increase in neurogenesis in the developing cortex, but results in a dramatic reduction in neurogenesis in the medial and lateral ganglionic eminences of the embryonic forebrain. The reduction in neurogenesis in GE derived neural precursors was accompanied by an increase in the production of immature astrocytes. We could further demonstrate that treatment with recombin ant BMP2 increased the production of astrocytes in neural precursors derived from GE, whereas no significant in crease in astrogliogenesis was detected in cortical neural precursor cells.

A co treatment with TSA and noggin, a BMP2 inhibitor, or with Alk3 ECD, a recombinant protein that contains the extracellular domain of the BMPR1A receptor, was able to restore the normal levels of neurons and astrocytes, compared to untreated control samples, demonstrating a direct connection between HDAC activ ity and BMP signaling. In order to investigate the sig naling pathways involved in the differentiation of GE derived neural precursors upon TSA and BMP2 treat ment, we performed gene expression profiling and protein analysis from BMP2 or TSA treated neural precursor cells derived from GE at different time points. Here, we show that BMP2 and TSA influence neurogenesis in a related manner. We demonstrate that in the early response to BMP2 and TSA treatment, different cohorts of functional gene groups are activated or repressed, although the downstream biological effects are closely related.

We fur ther characterized individual genes picked up by the microarrays at both mRNA and protein levels. Results In vitro differentiation of forebrain derived neurosphere cultures We used neurosphere cultures to generate a uniform population of neural precursors directly from the medial and lateral ganglionic eminences of E15. 5 C57BL/6 mice. After 7 days neurospheres were dissociated, plated out as a monolayer, and differentiated according to stan dard protocols. During differentiation FGF2 was withdrawn after 2. 5 days, whereas the treatment with TSA or BMP2 Brefeldin_A started 1. 5 days after plating. Cultures were allowed to differentiate for an add itional 4. 5 days after FGF2 withdrawal and then stained with immunocytofluorescence for standard markers indicating the birth of newborn neurons, astrocytes, and oligodendrocytes. As reported previously, both TSA as well as BMP2 treatment suppressed neurogenesis and boosted astrogliogenesis, as indicated by the BMP2 TSA relative number of TuJ1 positive neurons and GFAP positive astrocytes in the cultures. Simul taneous treatment with both TSA and BMP2 showed a similar effect.