Biology subjects

Plath, K.

Publications and source records attributed to Plath, K..

5 recordsLinked to original sources

bioRxiv2018

A single cell transcriptomic analysis of human neocortical development

Defining the number, proportion, or lineage of distinct cell types in the developing human brain is an important goal of modern brain research. We defined single cell transcriptomic profiles for 40,000 cells at mid-gestation to identify cell types in the developing human neocortex. We define expression profiles corresponding to all known major cell types at this developmental period and identify multiple transcription factors and co-factors expressed in specific cell types, providing an unprecedented resource for understanding human neocortical development including the first single-cell characterization of human subplate neurons. We characterize major developmental trajectories during early neurogenesis, showing that cell type differentiation occurs on a continuum that involves transitions that tie cell cycle progression with early cell fate decisions. We use these data to deconvolute regulatory networks and map neuropsychiatric disease genes to specific cell types, implicating dysregulation of specific cell types, as the mechanistic underpinnings of several neurodevelopmental disorders. Together these results provide an extensive catalog of cell types in human neocortex and extend our understanding of early cortical development, human brain evolution and the cellular basis of neuropsychiatric disease.\n\nOne Sentence SummaryComprehensive single cell transcriptomes in developing human cortex inform models of cell diversity, differentiation and disease risk.

neuroscience↗

bioRxiv2017

Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPS cells

Both human and mouse fibroblasts can be reprogrammed to pluripotency with Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription factors. While both systems generate pluripotency, human reprogramming takes considerably longer than mouse. To assess additional similarities and differences, we sought to compare the binding of the reprogramming factors between the two systems. In human fibroblasts, the OSK factors initially target many more closed chromatin sites compared to mouse. Despite this difference, the intra- and intergenic distribution of target sites, target genes, primary binding motifs, and combinatorial binding patterns between the reprogramming factors are largely shared. However, while many OSKM binding events in early mouse cell reprogramming occur in syntenic regions, only a limited number is conserved in human. In summary, these findings suggest similar general effects of OSKM binding across these two species, even though the detailed regulatory networks have diverged significantly.

genomics↗

bioRxiv2017

DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS)

Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale(1). Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome(16,19). Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq(12)). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

genomics↗

bioRxiv2017

Epigenetic resetting of human pluripotency

Much attention has focussed on conversion of human pluripotent stem cells (PSC) to a more naive developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 hours. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from primed PSC and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to the level in the ICM but is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of bi-allelic X-linked gene transcription indicates re-activation of the silenced X chromosome. On re-conversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naive cells.

developmental biology↗

bioRxiv2017

Loss of MECP2 leads to telomere dysfunction and neuronal stress

To determine the role for mutations of MECP2 in Rett Syndrome, we generated isogenic lines of human iPSCs (hiPSCs), neural progenitor cells (NPCs), and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal specific gene expression changes including induction of a Senescence Associated Secretory Phenotype (SASP) program. Patient derived Neurons made without MECP2 show signs of stress, including induction of p53, and senescence. The induction of p53 appeared to affect dendritic branching in Rett neurons, as p53 inhibition restored dendritic complexity. These disease-in-a-dish data suggest that loss of MECP2 can lead to dendritic defects due to an increase in aspects of neuronal aging.

developmental biology↗