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Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development

ABSTRACT: Human embryonic stem cells (hESCs) were specified as ventral telencephalic neuroectoderm (day 4) and then into medial ganglionic emininence (MGE)-like progenitors (day 15) and were subsequently differentiated into cortical interneuron (cIN)-like cells (day 25-35), by modification of previously published protocols. RNA-seq analysis at days 0, 4, 15, 25, and 35 defined transcriptome signatures for MGE and cIN cell identity. Further integration of these gene expression signatures with ChIP-seq for the NKX2-1 transcription factor in MGE-like progenitors defined NKX2-1 putative direct targets, including genes involved in both MGE specification and in several aspects of later cIN differentiation (migration, synaptic function). Among the NKX2-1 direct targets with MGE and cIN enriched expression was CHD2, a chromatin remodeling protein. Since CHD2 haploinsufficiency can cause epilepsy and/or autism, which can involve altered cIN development or function, we evaluated CHD2 requirements in these processes. Transcriptome changes were evaluated in CHD2 knockdown MGE-like progenitors at day 15, revealing diminished expression of genes involved in MGE specification and cIN differentiation including channel and synaptic genes implicated in epilepsy, while later cIN electrophysiological properties were also altered. We defined some shared cis-regulatory elements bound by both NKX2-1 and CHD2 and characterized their ability to cooperatively regulate cIN gene transcription through these elements. We used these data to construct regulatory networks underlying MGE specification and cIN differentiation and to define requirements for CHD2 and its ability to cofunction with NKX2-1 in this process.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99937 | GEO | 2017/11/30

SECONDARY ACCESSION(S): PRJNA390153

REPOSITORIES: GEO

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Project description:The Dlx homeodomain transcription factors are implicated in regulating the function of inhibitory GABAergic interneurons; therefore understanding their functions will provide insights into disorders such as epilepsy, mental retardation and autism. Identifying genes that are downstream of Dlx1/2 function and are relevant for the differentiation and survival of GABAergic interneurons. During embryonic development, cortical GABAergic interneurons are generated in the proliferative zone of the medial ganglionic eminence (MGE), from where they migrate to reach their final positions in the cortex. The differentiation of these interneuron precursors is dependent on Dlx genes, as shown by Dlx1/Dlx2 double mutants, which have a block in GABAergic cell differentiation and in cell migration. When interneuron progenitors are isolated from the mutant MGE and growth in culture, they are able to proceed along their differentiation program. However, mutant cells growth in vitro show defects in cell morphogenesis and increased cell apoptosis. We hypothesize that Dlx transcription factors regulate important aspects of GABAergic neuron differentiation such as the formation and growth of axon and dendrites, and the formation of inhibitory synapses. We generated E15.5 mouse embryos that are Dlx1/2 -/- or Dlx1/2 +/?. Genotype was confirmed by PCR. A total of 8 litters were used. For each experiment, we pooled tissue from at least 6 different embryos of the same genotype. We dissected the ventricular and subventricular zones of the MGE (rostral part). This area contains ~1 million of progenitor cells per embryo. We isolated total RNA using the Stratagene RNA Miniprep kit (these samples are called MGE+/ and MGE-/- in our proposal). In addition, we used the same area (ventricular and subventricular zones of the rostral MGE) to perform primary neuronal cultures. Cells were maintained 3 days in vitro. After that, we isolated total RNA using the Stratagene RNA Miniprep kit (samples called primary cells+/ and primary cells-/- in our proposal). We would like to perform gene expression comparison between: 1) MGE+/ and MGE-/-, and 2) primary cells+/ and primary cells-/-.

Project description:Aims: MiRNAs are post-transcriptional regulatory molecules with recognized roles in human heart development and disease. The purpose of this study was to define human miRNA expression profile in cardiac progenitors and early-differentiated cardiomyocytes and to determine if these miRNAs that are governing cardiogenesis is dependent on NKX2-5, a highly conserved pan-cardiac transcription factor. Methods: MiRNA expression profiles of pre-NKX2-5 mesoderm, cardiac progenitors and early cardiomyocytes derived from heterozygous HES3 NKX2-5eGFP/w and null NKX2-5eGFP/eGFP hESC lines were generated by small RNA sequencing in triplicates, using Illumina HiSeq. Reads between 17-28 base pairs were selected for sequence alignment to the human genome (hg19) using Bowtie2. FeatureCounts (Subread, R package) was used to count the number of reads mapping to miRNAs and analysis of statistically differentially expressed miRNA was carried out using Limma (Bioconductor) through the statistical language R. qRT–PCR validation was performed using Taqman MicroRNA Assays. Results: We identified 11 miRNAs that were differentially expressed between pre-NKX2-5 mesoderm and cardiac progenitors, and 112 differentially expressed miRNAs between cardiac progenitors and early cardiomyocytes. Four of which were validated with qRT–PCR, including canonical myogenic miRNAs such as MIR-1-1, -133A1 and 208A that were enriched in both the cardiac progenitor and early cardiomyocyte populations. Strikingly, deletion of NKX2-5 did not result in gross changes in cardiac miRNA profile either at progenitor or cardiomyocyte stage. Instead, non-hierarchical clustering and principal component analysis demonstrated that the different stages of differentiation (day 6 vs day 10) was a larger discriminator between samples than NKX2-5 genotype. Conclusion: Our study demonstrates the application of human embryonic stem cells as an in vitro model to investigate the role of miRNAs in human cardiac development. We conclude that while specific miRNAs have been identified in our study to have a role in the early stages cardiac cell fate specification, the majority of cardiac myogenic miRNA program is regulated separately from the highly conserved NKX2-5 -dependent gene network. This study provides a framework for further investigation of other key transcription factors of cardiac muscle development that might drive cardiomyogenic expression of miRNAs.

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Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development

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