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Bienko M.,Massachusetts Institute of Technology | Crosetto N.,Massachusetts Institute of Technology | Teytelman L.,Massachusetts Institute of Technology | Klemm S.,Massachusetts Institute of Technology | And 4 more authors.
Nature Methods | Year: 2013

We developed a cost-effective genome-scale PCR-based method for high-definition DNA FISH (HD-FISH). We visualized gene loci with diffraction-limited resolution, chromosomes as spot clusters and single genes together with transcripts by combining HD-FISH with single-molecule RNA FISH. We provide a database of over 4.3 million primer pairs targeting the human and mouse genomes that is readily usable for rapid and flexible generation of probes. © 2013 Nature America, Inc. All rights reserved. Source

Etemad B.,Hubrecht Institute KNAW Royal Netherlands Academy of Arts and science | Kops G.J.P.L.,Hubrecht Institute KNAW Royal Netherlands Academy of Arts and science | Kops G.J.P.L.,University Utrecht
Current Opinion in Cell Biology | Year: 2016

Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. © 2016 Elsevier Ltd. Source

Soh Y.Q.S.,Whitehead Institute For Biomedical Research | Soh Y.Q.S.,Massachusetts Institute of Technology | Junker J.P.,Massachusetts Institute of Technology | Junker J.P.,Hubrecht Institute KNAW Royal Netherlands Academy of Arts and science | And 11 more authors.
PLoS Genetics | Year: 2015

The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once. © 2015 Soh et al. Source

Grun D.,Hubrecht Institute KNAW Royal Netherlands Academy of Arts and science | Grun D.,University Utrecht | Grun D.,Max Planck Institute of Immunobiology and Epigenetics | Van Oudenaarden A.,Hubrecht Institute KNAW Royal Netherlands Academy of Arts and science | Van Oudenaarden A.,University Utrecht
Cell | Year: 2015

Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing. © 2015 Elsevier Inc. Source

Peterson K.A.,Harvard University | Peterson K.A.,University of Southern California | Nishi Y.,Harvard University | Nishi Y.,University of Southern California | And 23 more authors.
Genes and Development | Year: 2012

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a contextspecific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube. © 2012 by Cold Spring Harbor Laboratory Press. Source

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