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Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

Metzakopian E, Bouhali K, Alvarez-Saavedra M, Whitsett JA, Picketts DJ, Ang SL - Development (2015)

Bottom Line: Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity.Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons.Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Neurobiology, NIMR, The Ridgeway, London NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus

Overexpression of Smarca1 induces a higher efficiency of mDA neuron generation in vitro. (A) Immunostaining of day (d) 14 cells after monolayer differentiation as derived from rtTA Smarca1 epiblast stem cells (EpiSCs). (B) Quantification of the immunostaining in A, illustrating the percentage of DAPI-stained cells expressing Th in doxycycline (Dox)-treated and untreated cultures at d14 following monolayer differentiation. Mean±s.e.m. of three independent replicates. (C) The in vitro differentiation procedure. EpiSC monolayer cultures were exposed to the fibroblast growth factor inhibitor PD0325901 (PD) from d0 to d2. (D) qRT-PCR analyses of Nurr1, Aadc and Vmat2 in EpiSCs, d10 and d14 cultures after monolayer differentiation of rtTA Smarca1 EpiSCs with or without Dox treatment. Error bars indicate s.e.m. (B,D) *P<0.05 (two-tailed t-test).
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DEV115808F7: Overexpression of Smarca1 induces a higher efficiency of mDA neuron generation in vitro. (A) Immunostaining of day (d) 14 cells after monolayer differentiation as derived from rtTA Smarca1 epiblast stem cells (EpiSCs). (B) Quantification of the immunostaining in A, illustrating the percentage of DAPI-stained cells expressing Th in doxycycline (Dox)-treated and untreated cultures at d14 following monolayer differentiation. Mean±s.e.m. of three independent replicates. (C) The in vitro differentiation procedure. EpiSC monolayer cultures were exposed to the fibroblast growth factor inhibitor PD0325901 (PD) from d0 to d2. (D) qRT-PCR analyses of Nurr1, Aadc and Vmat2 in EpiSCs, d10 and d14 cultures after monolayer differentiation of rtTA Smarca1 EpiSCs with or without Dox treatment. Error bars indicate s.e.m. (B,D) *P<0.05 (two-tailed t-test).

Mentions: Given the possibility of redundant functions of different members of the SWI/SNF family that would mask the roles of any single member, we performed gain-of-function studies of Smarca1 function using a mouse epiblast stem cell differentiation protocol to generate mDA neurons (Jaeger et al., 2011). We first generated stably transfected ESCs carrying a doxycycline-inducible Smarca1 expression construct. These Smarca1 gain-of-function ESCs were differentiated first into epiblast cells. These cells were then grown in differentiation medium alone or in the presence of doxycycline so that Smarca1 expression was induced in postmitotic mDA neurons at day 5 of monolayer differentiation, and the cells were then cultured to day 14 to generate βIII-tubulin+ Th+ neurons (Fig. 7A-C). As expected, some Th+ neurons (37%) were observed in the parental or non-induced control cultures (Fig. 7A,B). By contrast, a higher proportion of Th+ neurons (62%) differentiated in monolayer cultures overexpressing Smarca1 (Fig. 7A,B). Most of the Th+ neurons were found in large clusters and had a mature neuronal phenotype (Fig. 7A). We also found by RT-qPCR that the expression of other mature mDA neuron markers, such as Nurr1, Aadc (Ddc) and Vmat2 (Slc18a2), was also enhanced in cultures with induced Smarca1 expression compared with control cultures (Fig. 7D).Fig. 7.


Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

Metzakopian E, Bouhali K, Alvarez-Saavedra M, Whitsett JA, Picketts DJ, Ang SL - Development (2015)

Overexpression of Smarca1 induces a higher efficiency of mDA neuron generation in vitro. (A) Immunostaining of day (d) 14 cells after monolayer differentiation as derived from rtTA Smarca1 epiblast stem cells (EpiSCs). (B) Quantification of the immunostaining in A, illustrating the percentage of DAPI-stained cells expressing Th in doxycycline (Dox)-treated and untreated cultures at d14 following monolayer differentiation. Mean±s.e.m. of three independent replicates. (C) The in vitro differentiation procedure. EpiSC monolayer cultures were exposed to the fibroblast growth factor inhibitor PD0325901 (PD) from d0 to d2. (D) qRT-PCR analyses of Nurr1, Aadc and Vmat2 in EpiSCs, d10 and d14 cultures after monolayer differentiation of rtTA Smarca1 EpiSCs with or without Dox treatment. Error bars indicate s.e.m. (B,D) *P<0.05 (two-tailed t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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DEV115808F7: Overexpression of Smarca1 induces a higher efficiency of mDA neuron generation in vitro. (A) Immunostaining of day (d) 14 cells after monolayer differentiation as derived from rtTA Smarca1 epiblast stem cells (EpiSCs). (B) Quantification of the immunostaining in A, illustrating the percentage of DAPI-stained cells expressing Th in doxycycline (Dox)-treated and untreated cultures at d14 following monolayer differentiation. Mean±s.e.m. of three independent replicates. (C) The in vitro differentiation procedure. EpiSC monolayer cultures were exposed to the fibroblast growth factor inhibitor PD0325901 (PD) from d0 to d2. (D) qRT-PCR analyses of Nurr1, Aadc and Vmat2 in EpiSCs, d10 and d14 cultures after monolayer differentiation of rtTA Smarca1 EpiSCs with or without Dox treatment. Error bars indicate s.e.m. (B,D) *P<0.05 (two-tailed t-test).
Mentions: Given the possibility of redundant functions of different members of the SWI/SNF family that would mask the roles of any single member, we performed gain-of-function studies of Smarca1 function using a mouse epiblast stem cell differentiation protocol to generate mDA neurons (Jaeger et al., 2011). We first generated stably transfected ESCs carrying a doxycycline-inducible Smarca1 expression construct. These Smarca1 gain-of-function ESCs were differentiated first into epiblast cells. These cells were then grown in differentiation medium alone or in the presence of doxycycline so that Smarca1 expression was induced in postmitotic mDA neurons at day 5 of monolayer differentiation, and the cells were then cultured to day 14 to generate βIII-tubulin+ Th+ neurons (Fig. 7A-C). As expected, some Th+ neurons (37%) were observed in the parental or non-induced control cultures (Fig. 7A,B). By contrast, a higher proportion of Th+ neurons (62%) differentiated in monolayer cultures overexpressing Smarca1 (Fig. 7A,B). Most of the Th+ neurons were found in large clusters and had a mature neuronal phenotype (Fig. 7A). We also found by RT-qPCR that the expression of other mature mDA neuron markers, such as Nurr1, Aadc (Ddc) and Vmat2 (Slc18a2), was also enhanced in cultures with induced Smarca1 expression compared with control cultures (Fig. 7D).Fig. 7.

Bottom Line: Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity.Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons.Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Neurobiology, NIMR, The Ridgeway, London NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus