Project description:We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/ sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate. Chromatin immunoprecipitation was performed using antibody against Pbx1/2/3/ (sc-888, Santa Cruz). Wild type E12.5 mouse whole cortex was used for the analysis.
Project description:E12.5 wild-type embryos were dissected to collect ventral midbrain regions. Samples were crosslinked 10min with 1% formaldehyde and processed for chromatin extraction and Chromatin Immunoprecipitation (ChIP) following the Millipore upstate protocol. Libraries were prepared using the illumina ChIP-Seq DNA Sample Prep Kit. ChIP-Seq libraries were sequenced on the Illumina GAIIx.
Project description:Investigations into the roles for Pbx1 and its transcriptional network in dopaminergic neuron development and Parkinson's Disease Three samples each from dorsal midbrain, forebrain, hindbrain, Alar plate, and ventral midbrain
Project description:We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/ sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate. Chromatin immunoprecipitation was performed using antibody against Pbx1/2/3 (sc-888, Santa Cruz). Wild type E15.5 mouse whole cortex was used for the analysis.
Project description:Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. We here explored pre-B-cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-Seq analysis uncovered PBX1 binding to a wide range of different genes. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass-spectrometry. Spatial transcriptomics revealed distinct temporal expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3, TCF4 and TCF12. RNA-seq upon Pbx1, Tcf3 and Tcf4 knockdown identified proliferation and differentiation associated genes as shared targets. Neuronal differentiation was reduced upon depletion of either factor, suggesting functional cooperation between PBX1 and TCF3/4. Notably, while physiological PBX1-TCF interactions have not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre B-cell line expressing TCF3 but lacking PBX1, upregulated leukemogenic genes including BLK and NOTCH3, arguing that functional PBX1-TCF cooperation likely extends to hematopoietic contexts. Our study hence uncovers a PBX1-TCF module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with implications for leukemia etiology.
Project description:To improve the standardization of cell therapies for Parkinson’s disease, methods for the selection and isolation of midbrain dopaminergic progenitors for transplantation are required. To facilitate this we established an expression profile for genes selectively expressed on transplantable midbrain dopaminergic progenitors using microarray analysis. Expression of GFP in the ventral mesencephalon of embryonic E12.5 Ngn2-GFP mice identifies a distinct sub-population of cells containing virtually all of the midbrain dopaminergic progenitors. Gene expression profiles from 3 biological replicates of FACS isolated GFP-positive cells from mouse Ngn2-GFP ventral mesencephalon were generated using microarrays. To reduce the likelihood of identifying transcripts from non-dopaminergic progenitors, 3 biological replicates of FACS isolated GFP-negative cells from mouse Lmx1a-GFP ventral mesencephalon (definitively non-dopaminergic) were used as a reference population.
Project description:We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/ sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.
Project description:We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/ sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.