TI - HDACs downregulate BMP2/4 signaling to promote GE neurogenesis in vitro . AB - To elucidate the molecular mechanism by which HDACs regulate neurogenesis , we investigated various developmental pathways known to control neurogenesis in vivo . We first examined the BMP2/4 signal transduction pathway . BMP2 , BMP4 , and their receptors are expressed in neurosphere cultures [14] and have been shown to promote astrocytic differentiation in vitro[14] and in vivo[15] . BMP2 is highly similar to BMP4 ( 93% amino acid identity ) , activates the same signaling pathway as BMP4 , is also expressed in the subventricular zone of embryonic striatum [14] , and like BMP4 was found to be expressed in differentiating neural stem cell cultures ( Fig 3C , D , 4B ) . As we expected HDACs to act upon a transcriptional level to affect neurogenesis and astrogliogenesis in GE , we performed quantitative , real-time RT-PCR upon mRNA extracted from TSA-treated neurosphere cultures , comparing gene expression levels to untreated cultures. 12 hours after treatment of 1.5 DIV cultures with TSA , the expression of the BMP2/4-specific receptors Bmpr1a , Bmpr 1b and Bmpr2 were unchanged in GE-derived cultures (Fig 3A) . In contrast , the expression of Bmp2 was upregulated dramatically in GE-derived cultures (Fig 3C) after 12 hours of TSA treatment , whereas Bmp4 was downregulated ( Fig 3C ) . We next examined the expression of the inhibitory factor Smad7[55] in our cultures . Smad7 expression was downregulated by TSA application at both concentrations ( Fig 3E ) . In addition , the expression of the astrogliogenesis-promoting transcription factors Stat1 and Stat3 was upregulated by TSA-treatment (Fig 3G) . Curiously , the expression of the neurogenesis-promoting transcription factor Ngn1 was also upregulated by TSA-treatment , while that of Ngn2 remained unaffected ( Fig 3I ) . All of these effects showed a TSA dose-dependence ( Fig 3C , E , G , I ) . In order to test the relevance of the transcriptional control of Bmp2 and Smad7 gene expression by HDACs , we decided to influence the BMP2/4 signaling pathway by boosting or inhibiting the extracellular signal . Recombinant BMP2 was applied to differentiating neurosphere-derived cultures one day before the withdrawal of bFGF at concentrations ranging from 10-100 ng/ml . This resulted in a dramatic effect , in that over 50% of the cells differentiated into astrocytes , as previously reported ( Fig 4C , D ; [14] ) . The GFAP+ cells were clustered together in what resembled astrocytic "islands" ( Fig 4C , panel 7 ) . We also observed similar clusters of GFAP+ cells in cultures in which 10 nM TSA was withdrawn after 5 days of treatment and then cultured for an additional 3 to 4 days , for a total of 10-11 days ( Fig 4C , panel 8 ) . In order to test the involvement of endogenous BMP signaling in normal and TSA-treated cultures , cells were treated with two different BMP2/4 inhibitors . Alk3-ECD is a recombinant protein consisting of the extracellular domain of the BMPR1A receptor that can bind to BMP2 and -4 with high affinity , preventing them from binding to the endogenous BMPR1A receptor [65] . Noggin is a secreted polypeptide that binds to BMPs and prevent their binding to and activation of their receptors [66] . Each inhibitor was added for 24 hours before bFGF withdrawal , with or without 10 nM TSA , and then removed when bFGF was withdrawn . The cultures were analyzed 4.5 days later , at 7 DIV . The use of either Alk3-ECD or noggin ( each at a concentration of 250 ng/ml ) alone did not affect neurogenesis ( Fig 4A , D ) or astrocyte production ( Fig 4C , D ) . However , addition of either BMP2/4 inhibitor in combination with 10 nM TSA completely restored normal levels of neurogenesis ( Fig 4A , D ) . In addition , the production of astrocytes was reduced to levels seen in untreated cells ( Fig 4C , D ) . Longer treatment of cultures with these reagents , starting 24 hours before bFGF withdrawal but continuing for an additional 4.5 days , produced similar results ( Neurogenesis : . . . . . . . Control : . . . . . . 31.1+-0.7% ; TSA : . . . . . . . 8.2+-1.1% ; Alk3-ECD : . . . . . . . 30.4+-1.1% ; Alk-3 & TSA : . . . . . . . 27.9+-1.4% ; Noggin : . . . . . . . 30.0+-3.9% ; Noggin & TSA : . . . . . . . 24.8+-2.3% , n = 2 , reagent concentrations as above ) . In order to investigate the effect of TSA upon neural stem cell cultures , we next examined the activation of the BMP2/4 signaling pathway . Binding of BMP2/4 to their cognate receptors results in the PHOSphorylation of two adjacent serine residues in the Smad1/5/8 proteins , followed by their translocation to the nucleus where they act as transcriptional regulators of BMP2/4-responsive genes [67] . We examined the cultures with an antibody that specifically recognizes Smad1/5/8 proteins PHOSphorylated at these serine residues , staining cultures 1 , 4 , 12 , and 24 hours after reagents were added ( Fig 4E ) . Addition of 10 ng/ml BMP2 to cultures 24 hours before bFGF withdrawal caused an accumulation of PHOSphorylated Smad1/5/8 in the nucleus after just 1 hour of treatment ( Fig 4E ) . This localization was sustained at 4 hours , whereas by 12 and 24 hours it was no longer detectable . A nuclear localization of PHOSphorylated Smad1/5/8 became visible after 12 hours of treatment with 50 nM TSA and after 24 hours of treatment with 10 nM TSA (Fig 4E) . This delay in Smad1/5/8 nuclear localization compared to BMP2 application may reflect the need to induce a change in transcription patterns of genes involved in BMP signaling , such as Bmp2 (Fig 3C) itself or Smad7 (Fig 3E) . Additionally , we investigated two other pathways that are known to regulate neurogenesis in mammals and that had been implicated in the inhibition of retinal neurogenesis in a mutation in the zebrafish hdac1 gene [46] . Examination by Western blot of the protein levels of the intracellular domain of Notch1 , representing the cleaved , activated domain [68] , revealed no change in its expression upon a 7-day treatment with 10 nM TSA , comparing cultures at 1 , 2.5 , 3.5 , and 7 DIV (Fig S4A) . To examine a possible involvement of the Wnt signaling pathway , we treated neurosphere-derived cultures with inhibitors of Wnt signaling , Dkk1 ( 05 ug/ml ) and sFRP2 ( 02 ug/ml ) . These inhibitors were added 24 hours before bFGF withdrawal , and neurogenesis examined after a further 4.5 days of culture . In the presence of the two inhibitors , no changes were seen in neurogenesis , in comparison with no TSA or with 10 nM TSA treatment , respectively ( Fig S4B ) . Although neither of these experiments definitively rule out the involvement of Notch or Wnt signaling in HDAC -mediated neurogenesis , the full complementation of neurogenesis seen with BMP2/4 inhibitors in combination with TSA treatment suggests that this is the most important neurogenic pathway controlled by HDACs .